Carbon Nanotube Compositions and Methods of Use Thereof

ABSTRACT

Carbon nanotube (CNT)-based compositions for activating cellular immune responses are provided. The CNTs function as high surface area scaffolds for the attachment of T cell ligands and/or antigens. The CNT compositions function as artificial antigen-presenting cells (aAPCs) or as modular vaccines. The disclosed CNT aAPCs are efficient at activating T cells and may be used to activate T cells ex vivo or in vivo for adoptive or active immunotherapy.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 12/933,223,which is the National Stage of International Application No.PCT/US09/37727 filed Mar. 19, 2009, which claims priority to and benefitof U.S. Provisional Patent Application No. 61/037,798, filed on Mar. 19,2008, the contents of which are incorporated by reference in theirentirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support awarded by the NationalScience Foundation under Career Award Number 0747577 to Tarek M. Fahmy.The United States government has certain rights in this invention.

FIELD OF THE INVENTION

The present disclosure generally relates to the field of carbon nanotubecompositions and methods for making and using these compositions.

BACKGROUND OF THE INVENTION

T cells are central players in initiating and maintaining immuneresponses. An important goal of successful immunotherapy is thestimulation of T cell immune responses against targets of interest suchas tumors. This can be accomplished in two ways: 1) through immunizationwith tumor antigens or 2) by isolation of T cells specific to tumorantigens, and expansion of this population outside the body followed byre-transfer into the patient (adoptive transfer immunotherapy).

Preventative vaccines have eliminated smallpox and nearly eliminatedpolio, two of the worst global infectious diseases. By contrast vaccinesfor many other infectious diseases, such as malaria and HIV, whichinvolve intracellular pathogens, are poorly developed or simplyunavailable. The lack of such vaccines will result in two millionunnecessary deaths each year in many parts of the world. Althougheconomic factors play a role, there are a number of significantscientific challenges that have limited the development of vaccines fordeadly diseases. First, few if any approaches are available thatefficiently prime cell-mediated immunity by direct intracellulardelivery of an antigen. Second, ‘tunable’ adjuvants, that is, adjuvantsthat can be engineered to optimize the magnitude and direction of animmune response, have not been developed. Third, the general requirementfor parenteral (i.e. subcutaneous or intramuscular injection)administration of vaccines, a situation that has made it difficult todeploy vaccines in underdeveloped countries where medical supportsystems, resources, and even refrigeration are limited. Finally, thereis a lack of a general approach to designing oral vaccines targeted toboth systemic and mucosal immunity; oral vaccines are significantly lessexpensive to administer and transport. Thus, there is a critical needfor safe and stable vaccine systems that would address these factors.

Some of the most encouraging data regarding immunotherapy come fromstudies employing adoptive transfer of tumor reactive T cells. AdoptiveT cell transfer is an elegant approach to the treatment of infectiousand malignant diseases. This therapeutic method involves the ex vivoexpansion of T cells, which may be infused into patients to bolster thenatural immune response. For example, expanded tumor-specific T cellshave been shown to strengthen patient's immune responses to melanoma byinfiltrating the tumor site and inducing tumor shrinkage. Researchershave also demonstrated that the adoptive transfer of T cells is a viabletherapeutic approach to treating Epstein-Barr virus (EBV) as well ashuman immunodeficiency virus (HIV)-related infections. Thus, adoptive Tcell transfer has potential applications in the treatment of bothinfectious diseases and cancer.

Despite the successes of these studies, adoptive T cell transfer byclonal expansion is not clinically viable since it does not consistentlygenerate therapeutic numbers of T cells. This shortcoming has promptedthe development of an alternative techniques for ex vivo T cellexpansion, using artificial antigen presentation to T cells (Prakken, etal., Nat. Med., 6(12):1406-10 (2000); Oelke, et al., Nat. Med.,9(5):619-24 (2003); Kim, et al., Nat. Biotechn., 22:403-10 (2004)). Thedevelopment of artificial APCs (aAPCs) is a new effort to generate areproducible, “off-the shelf” means of stimulating and expanding Tcells. Several types of aAPCs have been developed, including nonspecificbead-based systems that are currently used in many research laboratoriesto sustain the long-term expansion of CD8⁺ T cells (Oelke, et al., Nat.Med., 9(5):619-24 (2003); Kim, et al., Nat. Biotechn., 22:403-10(2004)).

Specific expansion of T cells outside the body depends however onefficient methods for displaying protein ligands that stimulate thosecells. Ultimately, T cell stimulus intensity depends on the density ofbound receptors in the contact area with a surface (Andersen, et al., J.Biol. Chem., 276(52):49125-32 (2001); Gonzalez, et al., Proc. Natl.Acad. Sci. U.S.A., 102(13):4824-9 (2005)). Regions with a high densityof T cell antigen receptors have been termed activated clusters becausethey are critical for T cell stimulation (Grakoui, et al., Science,285(5425:221-7 (1999); Monks, et al., Nature, 395(6697):82-6 (1998)).The presence of such high density clusters has also been shown toaccelerate T cell activation (Gonzalez, et al., Proc. Natl. Acad. Sci.U.S.A., 102(13):4824-9 (2005)). In the lymph node, the primary site forT cell stimulation, antigen presenting cells are thought to concentratethe presentation of T cell stimuli by trafficking in a densearchitectural scaffolding in close proximity to T cells.

It is therefore an object of the invention to provide compositions thatprovide for high density presentation of ligands to T cell surfacereceptors.

It is another object of the invention to provide modular vaccine systemswhich provide for flexible addition of antigens and other elements.

It is another object of the invention to provide methods for activatingT cells in vivo and ex vivo using compositions that provide high densityligand presentation.

It is yet another object of the invention to provide methods foractivating and expanding T cells in vivo or ex vivo using compositionsthat provide high density presentation of T cell ligands.

It is still another object of the invention to provide methods foractive and adoptive immunotherapy of diseases and disorders usingcompositions that provide high density presentation of T cell-activatingligands.

SUMMARY OF THE INVENTION

Carbon nanotube (CNT)-based compositions for activating cellular immuneresponses are provided. The CNTs function as high surface area scaffoldsfor the attachment of T cell ligands and/or antigens. CNTs may befabricated using any suitable method. CNTs may be single-walled carbonnanotubes (SWNTs) or multi-walled carbon nanotubes (MWNTs). Proteins maybe either covalently or non-covalently attached to the CNTs. The surfacearea of CNTs may be adjusted by chemical means, such as treatment withacid, prior to attachment of proteins.

CNT compositions that function as artificial antigen-presenting cells(aAPCs) include ligands attached to CNTs that bind to cell surfacereceptors on T cells, including at least one species of ligand thatbinds to and activates the T cell receptor. T cell receptor activatorsmay be antigen-specific or polyclonal T cell receptor activators.Suitable antigen-specific T cell receptor activators include antigensbound to MHC/HLA molecules. Exemplary antigens include, but are notlimited to, viral antigens, bacterial antigens, parasite antigens,allergens and environmental antigens, tumor antigens or autoantigens.Suitable polyclonal T cell receptor activators include, but are notlimited to, antibodies that crosslink the T cell receptor. CNT aAPCs mayalso include one or more costimulatory or T cell adhesion molecules tofacilitate T cell attachment to the aAPCs and T cell activation.

CNT compositions that function as modular vaccines do not directlyactivate T cells by binding to T cell surface receptors, but rather,facilitate the delivery of antigens to natural APCs in vivo. The modularCNT vaccine compositions include antigens and may further includeelements that facilitate antigen uptake by APCs, or APC activation.Suitable additional elements include adjuvants, dendritic cellrecognition elements, epithelial cell recognition elements, or moleculeswhich protect the vaccine compositions from degradation in low-pHenvironments.

Methods of using the disclosed CNT aAPCs and CNT vaccine compositions toactivate T cells are provided. The CNT aAPC compositions are extremelyefficient activators of T cells. The examples demonstrate that anti-CD3antibodies adsorbed onto SWNT bundles stimulate cells more efficientlythan equivalent concentrations of soluble anti-CD3 antibodies.Furthermore, SWNT bundles bound to anti-CD3 antibodies are moreefficient at activating T cells than other high surface areacompositions, such as activated carbon and polystyrene and C60nanoparticles, even when normalized by surface area. This indicates thatSWNTs possess unique properties in addition to their high surface areathat make them ideally suited to function as scaffolds for aAPCs andmake them superior to existing aAPC scaffolds.

Methods for using the CNT aAPCs for ex vivo activation of T cells foradoptive immunotherapy are provided. The methods include isolating apopulation of T cells from a subject to be treated, activating the Tcells with the CNT aAPCs, expanding the T cells, and administering the Tcells to the subject to be treated in an amount effective to induce animmune response.

Methods for adoptive immunotherapy of conditions associated withoveractivation of the immune system by ex vivo activation of regulatoryT cells are also provided.

Methods for active immunotherapy using the disclosed CNT aAPCs or CNTvaccine compositions are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic showing activation of T cells using single-wallednanotube (SWNT) scaffolds with adsorbed anti-CD3 antibodies (Ab). Thefirst step is adsorption of anti-CD3 antibodies onto the SWNT scaffold.Following washing, the anti-CD3-adsorbed SWNTs (SWNT+Ab) are incubatedwith T cells and the amount of T cell activation can be measured bydetermining the amount of cytokines, such as 11-2, released by the Tcells.

FIG. 2 is a schematic showing the design of carbon nanotube particulates(CNPs) for multivalent antigen presentation, paracrine delivery ofcytokine, and T cell enrichment. The first step involves bundlingneutravidin functionalized CNTs. Stochiometric amounts of biotinylated Tcell antigens are then added to be presented on the CNT surface. PLGAnanoparticles loaded with magnetite and IL-2 are bound to the antigenpresenting CNTs surface to yield the CNPs.

FIG. 3 is a schematic highlighting three properties of the engineeredCNP platform: multivalent antigen presentation, paracrine release ofIL-2, and magnetic separation/enrichment of CNPs from T cells.

FIG. 4 is a schematic showing the work flow for a T cell stimulationprocess and cell separation using CNPs; OT-1 CD8+ T cells were purifiedfrom splenocytes, and incubated with CNPs for three days; activated Tcells were then magnetically separated from CNPs, collected, andinjected peritumorally in B6 mice, which were previously inoculated withthe B16 tumor for ten days.

FIG. 5 is a line graph showing the total surface area of SWNTs that wereeither untreated (-♦-), treated with HNO₃ (-⋄-), or treated with HNO₃and then LiBH₄ (-∘-). Total surface area was estimated using nitrogenphysisorption and Brunauer-Emmett-Teller analysis. Data are presented asthe quantity of adsorbed gas as a function of the ratio of equilibriumto saturation pressure.

FIG. 6 is a line graph showing the amount of adsorption of a modelprotein, bovine serum albumin (BSA), to SWNTs that were either untreated(-∘-), treated with HNO₃ (-⋄-), or treated with HNO₃ and then LiBH₄(-▴-). The data are presented as the amount of BSA loaded onto SWNTsBSA/mg SWNT) as a function of the amount of BSA added to the SWNTs (μgBSA/mg SWNT).

FIG. 7 is a line graph showing the viability of T cells that were eitheruntreated or incubated with 2.5% sodium azide or increasingconcentrations of SWNTs. Data are expressed as the percent viability ofthe T cells as a function of the concentration of added SWNTs.

FIG. 8 is a line graph showing the activation of T cells by variousstimuli. T cells were activated either by HNO₃/LiBH₄ treated SWNTs withadsorbed anti-CD3 (-⋄-), plate-bound anti-CD3 (-Δ-), soluble anti-CD3(-∘-), or blank HNO₃/LiBH₄ treated SWNTs (-♦-). T cell activation wasdetermined by measuring the secretion of IL-2 and data are expressed asthe amount of IL-2 secreted (pg/ml) as a function of the concentrationof added anti-CD3 (μg/ml).

FIG. 9 is a line graph showing the activation of T cells by variousstimuli. T cells were activated either by soluble anti-CD3 (-Δ-) or byanti-CD3 adsorbed onto 3 M HNO₃/LiBH₄ treated SWNTs (-⋄-), 3 M HNO₃treated SWNTs (-▴-) or untreated SWNTs. T cell activation was determinedby measuring the secretion of IL-2 and data are expressed as the amountof IL-2 secreted (pg/ml) as a function of the concentration of addedanti-CD3 (μg/ml).

FIG. 10 is a line graph showing the activation of T cells by variousstimuli. T cells were activated either by anti-CD3-adsorbed at 100 μg/ml(-⋄-), 50 μg/ml (-

-), 25 μg/ml (-▴-) or 12.5 μg/ml (-♦-). All groups were treated with thesame amount of anti-CD3 during adsorption. T cell activation wasdetermined by measuring the secretion of IL-2 and data are expressed asthe amount of IL-2 secreted (pg/ml) as a function of the concentrationof added anti-CD3 (μg/ml).

FIG. 11 is a line graph showing the activation of T cells by variousstimuli. T cells were activated by anti-CD3-adsorbed adsorbed ontoeither 3 M HNO₃/LiBH₄ treated SWNTs (-∘-), untreated activated carbon(A.C.) (-▾-), 3 M HNO₃ treated A.C. (-▴-), or 3 M HNO₃/LiBH₄ treatedA.C. (-♦-). All materials were used at a concentration of 50 μg/ml, andinitially loaded with the same concentration of anti-CD3. T cellactivation was determined by measuring the secretion of IL-2 and dataare expressed as the amount of IL-2 secreted (pg/ml) as a function ofthe concentration of added anti-CD3 (μg/ml).

FIG. 12 is a line graph showing the activation of T cells by multiplehigh surface area materials. T cells were activated by anti-CD3 adsorbedonto either 3 M HNO₃/LiBH₄ treated SWNTs (-∘-), 3 M HNO₃ treated SWNTs(--), untreated SWNTs (-♦-), untreated A.C. (-⋄-), 3 M HNO₃ treatedA.C. (-

-), C60 nanoparticles (-X-), hydroxylated C60 nanoparticles (C60-OH) (-

-), hydroxylated 200 nm polysterene (PS) beads (PS-OH) (-Δ-), orcarboxylated 200 nm PS beads PS-COOH (-∇-). T cell activation wasdetermined by measuring the secretion of IL-2 and data are expressed asthe amount of IL-2 secreted (pg/ml) as a function of the concentrationof added anti-CD3 (μg/ml) normalized to the antibody per materialsurface area.

FIG. 13 is a line graph showing activation of spleenocytes obtained frommice vaccinated subcutaneously with ovalbumin (OVA) alone (-♦-), OVAadsorbed onto SWNTs (-∘-) OVA adsorbed onto alum (-Δ-), or phosphatebuffered saline (PBS) (-

-), as a control. Spleenocytes obtained from the mice were activatedwith OVA at the indicated concentration. Spleenocyte activation wasdetermined by measuring secretion of IL-2 and data are expressed as theamount of IL-2 secreted (pg/ml) as a function of the concentration ofadded OVA (μg/ml).

FIG. 14 is a graph showing activation of spleenocytes obtained from micevaccinated intraperitoneally or orally with OVA, OVA adsorbed ontoSWNTs, OVA adsorbed onto alum, or PBS, as a control. Spleenocytesobtained from the mice were activated with OVA at 1 mg/ml. Spleenocyteactivation was determined by measuring secretion of IL-2 and data areexpressed as the amount of secreted IL-2 (pg/ml).

FIG. 15 is a bar graph showing the size distribution of bundled CNTmicroparticles extracted from SEM images and using NIH ImageJ software(n=142 particles).

FIG. 16 is plot of the size distribution of the magnetite and CL-2loaded PLGA nanoparticles using nanoparticle tracking analysis.

FIG. 17 is graph showing the encapsulated IL-2 (solid grey circles, leftaxis) and iron (empty circles, right axis) release from magnetite andCL-2 loaded PLGA nanoparticles over a 1-week period. The results aremean values from three independent experiments with error barsrepresenting ±SEM.

FIG. 18 is a graph showing OT-1 CD8+ T cell expansion measured usingcoulter-counter during a two-week period. The results are mean valuesfrom three independent experiments with error bars representing ±SD.

FIG. 19 is a bar graph showing IFN-γ release from OT-1 CD8+ T cellsmeasured at each time point during a two-week period cell expansion. Theresults are mean values from three independent experiments with errorbars representing ±SEM. (*) represents a p-value <0.05, (**) representsa p-value <0.01, and (***) represents a p-value <0.0001.

FIG. 20 is a graph showing the effect of CNPs on CD8+ T cell phenotypeand cytolytic activity summarizing the percentage of CD8+/CD27+ T cellsby group as a function of time. Gating was performed on live T cells.

FIG. 21 is a graph showing the effect of CNPs on CD8+ T cell phenotypeand cytolytic activity summarizing the percentage of CD25+/CD69+ T cellsby group as a function of time. Gating was performed on live T cells.

FIG. 22 is a graph showing the effect of CNPs on CD8+ T cell phenotypeand cytolytic activity summarizing the percentage of CD44+/CD8+ T cellsby group as a function of time. Gating was performed on live T cells.

FIG. 23 is a bar graph showing the expression of intracellulargranzyme-B at day 3 in OT-1 CD8+ T cells activated by CNPs vs.established controls. (***) represents a p-value <0.0001.

FIG. 24 is a bar graph showing cytotoxic activity of OT-1 CD8+ T cellstowards B16 cells presenting MHC-I in the context of OVA. Error barsrepresent the means±SEM. All data are representative of at least threeindependent experiments. (*) represents a p-value <0.05 FIG. 25 is a bargraph showing the expression of intracellular granzyme-B at day 3 inOT-1 CD8+ T cells activated by CNPs vs. established controls containinga thousand fold higher exogenous IL-2 concentrations.

FIG. 26 is a bar graph showing cytotoxic activity of OT-1 CD8+ T cellstowards B16 cells presenting MHC-I in the context of OVA vs. controlscontaining a thousand fold higher exogenous IL-2 concentrations. Errorbars represent the means±SEM. All data are representative of at leastthree independent experiments.

FIG. 27 is a bar graph showing the normalized tumor volume in C57BL/6mice previously inoculated with B16F10-OVA for ten days, and injectedperitumorally with 1.106 OT-1 CD8+ T cells. T cells were previouslyactivated by CNPs or established controls containing a thousand foldhigher exogenous IL-2 concentrations. Tumor volumes were normalized tovolumes measured at day 10 for each group. The results are mean valuesfrom 6 mice per group, with error bars representing ±SEM. (*) representsp-value <0.05.

FIG. 28 is a graph showing measured tumor mass in C57BL/6 micepreviously inoculated with B16F10-OVA for ten days, and injectedperitumorally with 1.106 OT-1 CD8+ T cells. T cells were previouslyactivated by CNPs or established controls containing a thousand foldhigher exogenous IL-2 concentrations. Measurement of tumor mass for eachgroup. Mice were euthanized directly before tumor mass determination;error bars represent ±SEM.

FIG. 29 is a bar graph showing the absolute number of activated T cellspresent in the tumor from C57BL/6 mice previously inoculated withB16F10-OVA for ten days, and injected peritumorally with 1×10⁶ OT-1 CD8+T cells per mouse; the T cells were previously activated by CNPs orestablished controls containing a thousand fold higher exogenous IL-2concentrations. The results are mean values from 3 tumors per group;error bars represent ±SEM. (*) represents p-value <0.05.

FIG. 30 is a bar graph showing the absolute number ofCD44+/CD62L-effector T cells present in the tumor from C57BL/6 micepreviously inoculated with B16F10-OVA for ten days, and injectedperitumorally with 1×10⁶ OT-1 CD8+ T cells per mouse; the T cells werepreviously activated by CNPs or established controls containing athousand fold higher exogenous IL-2 concentrations. The results are meanvalues from 3 tumors per group; error bars represent ±SEM. (*)represents p-value <0.05.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

An “antigen” is defined herein as a molecule which contains one or moreepitopes that will stimulate a host's immune system to make a cellularantigen-specific immune response, and/or a humoral antibody response.Antigens can be peptides, proteins, polysaccharides, saccharides,lipids, nucleic acids, and combinations thereof. The antigen can bederived from a virus, bacterium, parasite, plant, protozoan, fungus,tissue or transformed cell such as a cancer or leukemic cell and can bea whole cell or immunogenic component thereof, e.g., cell wallcomponents. An antigen may be an oligonucleotide or polynucleotide whichexpresses an antigen. Antigens can be natural or synthetic antigens, forexample, haptens, polyepitopes, flanking epitopes, and other recombinantor synthetically derived antigens (Bergmann, et al., Eur. J. Immunol.,23:2777-2781 (1993); Bergmann, et al., J. Immunol., 157:3242-3249(1996); Suhrbier, Immunol. and Cell Biol., 75:402-408 (1997).

A “tumor-specific antigen” is defined herein as an antigen that isunique to tumor cells and does not occur in or on other cells in thebody.

A “tumor-associated antigen” is defined herein as an antigen that is notunique to a tumor cell and is also expressed in or on a normal cellunder conditions that fail to induce an immune response to the antigen.

As used herein, the term “isolated” describes a compound of interest(e.g., either a polynucleotide or a polypeptide) that is in anenvironment different from that in which the compound naturally occurs,e.g., separated from its natural milieu such as by concentrating apeptide to a concentration at which it is not found in nature.“Isolated” includes compounds that are within samples that aresubstantially enriched for the compound of interest and/or in which thecompound of interest is partially or substantially purified.

As used herein, the term “polypeptide” refers to a chain of amino acidsof any length, regardless of modification (e.g., phosphorylation orglycosylation).

As used herein, a “variant” polypeptide contains at least one amino acidsequence alteration (addition, deletion, substitution, preferablyconservative i.e., not substantially changing the function except inmagnitude) as compared to the amino acid sequence of the correspondingwild-type polypeptide.

As used herein, an “amino acid sequence alteration” can be, for example,a substitution, a deletion, or an insertion of one or more amino acids.

As used herein, a “fragment” of a polypeptide refers to any subset ofthe polypeptide that is a shorter polypeptide of the full lengthprotein. Generally, fragments will be five or more amino acids inlength.

As used herein, “conservative” amino acid substitutions aresubstitutions wherein the substituted amino acid has similar structuralor chemical properties.

As used herein, “non-conservative” amino acid substitutions are those inwhich the charge, hydrophobicity, or bulk of the substituted amino acidis significantly altered.

As used herein, “isolated nucleic acid” refers to a nucleic acid that isseparated from other nucleic acid molecules that are present in amammalian genome, including nucleic acids that normally flank one orboth sides of the nucleic acid in a mammalian genome. As used hereinwith respect to nucleic acids, the term “isolated” includes anynon-naturally-occurring nucleic acid sequence, since suchnon-naturally-occurring sequences are not found in nature and do nothave immediately contiguous sequences in a naturally-occurring genome.

As used herein, the term “host cell” refers to prokaryotic andeukaryotic cells into which a recombinant expression vector can beintroduced.

As used herein, “transformed” and “transfected” encompass theintroduction of a nucleic acid (e.g. a vector) into a cell by a numberof techniques known in the art.

As used herein, the phrase that a molecule “specifically binds” to atarget refers to a binding reaction which is determinative of thepresence of the molecule in the presence of a heterogeneous populationof other biologics. Thus, under designated immunoassay conditions, aspecified molecule binds preferentially to a particular target and doesnot bind in a significant amount to other biologics present in thesample. Specific binding of an antibody to a target under suchconditions requires the antibody be selected for its specificity to thetarget. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectmonoclonal antibodies specifically immunoreactive with a protein. See,e.g., Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Publications, New York, for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity. Specific binding between two entities means anaffinity of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M⁻¹. Affinities greaterthan 10⁸M⁻¹ are preferred.

As used herein, the terms “antibody” or “immunoglobulin” include intactantibodies and binding fragments thereof. Typically, fragments competewith the intact antibody from which they were derived for specificbinding to an antigen fragment including separate heavy chains, lightchains Fab, Fab′ F(ab′)2, Fabc, and Fv. Fragments are produced byrecombinant DNA techniques, or by enzymatic or chemical separation ofintact immunoglobulins. The term “antibody” also includes one or moreimmunoglobulin chains that are chemically conjugated to, or expressedas, fusion proteins with other proteins. The term “antibody” alsoincludes bispecific antibody. A bispecific or bifunctional antibody isan artificial hybrid antibody having two different heavy/light chainpairs and two different binding sites. Bispecific antibodies can beproduced by a variety of methods including fusion of hybridomas orlinking of Fab′ fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp.Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol., 148,1547-1553 (1992).

As used herein, the terms “epitope” or “antigenic determinant” refer toa site on an antigen to which B and/or T cells respond. B-cell epitopescan be formed both from contiguous amino acids or noncontiguous aminoacids juxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 amino acids, in a uniquespatial conformation. Methods of determining spatial conformation ofepitopes include, for example, x-ray crystallography and 2-dimensionalnuclear magnetic resonance. See, e.g., Epitope Mapping Protocols inMethods in Molecular Biology, Vol. 66, Glenn E. Morris, Ed. (1996).Antibodies that recognize the same epitope can be identified in a simpleimmunoassay showing the ability of one antibody to block the binding ofanother antibody to a target antigen. T-cells recognize continuousepitopes of about nine amino acids for CD8 cells or about 13-15 aminoacids for CD4 cells. T cells that recognize the epitope can beidentified by in vitro assays that measure antigen-dependentproliferation, as determined by ³H-thymidine incorporation by primed Tcells in response to an epitope (Burke, et al., J. Inf. Dis.,170:1110-19 (1994)), by antigen-dependent killing (cytotoxic Tlymphocyte assay, Tigges, et al., J. Immunol., 156:3901-3910) or bycytokine secretion.

As used herein, the terms “immunologic”, “immunological” or “immune”response is the development of a humoral (antibody mediated) and/or acellular (mediated by antigen-specific T cells or their secretionproducts) response directed against an antigen. Such a response can bean active response induced by administration of immunogen or a passiveresponse induced by administration of antibody or primed T-cells. Acellular immune response is elicited by the presentation of polypeptideepitopes in association with Class I or Class II MHC molecules toactivate antigen-specific CD4⁺ T helper cells and/or CD8⁺ cytotoxic Tcells. The response may also involve activation of monocytes,macrophages, NK cells, basophils, dendritic cells, astrocytes, microgliacells, eosinophils or other components of innate immunity. The presenceof a cell-mediated immunological response can be determined byproliferation assays (CD4⁺ T cells) or CTL (cytotoxic T lymphocyte)assays. The relative contributions of humoral and cellular responses tothe protective or therapeutic effect of an immunogen can bedistinguished by separately isolating antibodies and T-cells from animmunized syngeneic animal and measuring protective or therapeuticeffect in a second subject.

As used herein, a “costimulatory polypeptide” or a “costimulatorymolecule” is a polypeptide that, upon interaction with a cell-surfacemolecule on T cells, enhances T cell responses, enhances proliferationof T cells, enhances production and/or secretion of cytokines by Tcells, stimulates differentiation and effector functions of T cells orpromotes survival of T cells relative to T cells not contacted with acostimulatory peptide.

II. Carbon Nanotube Compositions

Compositions based on carbon nanotubes (CNTs) for activating cellularimmune responses are disclosed. In one embodiment, the CNT compositionscontain ligands for T cell surface receptors and function as artificialantigen-presenting cell (aAPCs) to directly activate T cells either invivo or ex vivo. In another embodiment, the CNT compositions containantigens and are used as modular vaccine systems to deliver the antigento professional APCs to activate T cells in vivo.

A. Carbon Nanotubes

The compositions include carbon nanotubes (CNTs) as high surface areascaffolds for the attachment or ligands and/or antigens. A carbonnanotube is a crystalline carbon with a structure in which a thin layerof graphite crystal is rolled-up into the shape of a cylinder. CNTs areformed of carbons atoms in the form of a graphene structure, which is aflat or curved layer formed by arranging six-membered rings of carbonatoms in a honeycomb. A carbon nanotube is a cylindrical structure inwhich such a layer is rolled-up in one direction. In general, those witha diameter of several nanometers to several ten of nanometers and alength of several ten times to not less than several thousand timeslonger than its diameter are called “carbon nanotubes”.

CNTs that form the scaffold may be either single-walled CNTs (SWNTs) ormulti-walled CNTs (MWNTs). In a preferred embodiment, the compositionscontain SWNTs. SWNTs are formed by a single graphene layer rolled-up inthe shape of a cylinder. MWNTs are formed by two or more graphene layersrolled-up in the shape of a cylinder. Single-walled carbon nanotubes mayassume three types of shapes, termed “armchair”, “zigzag”, and “chiral”,depending on how the six-membered rings are arranged.

SWNTs have applications ranging from electronics (Ouyang, et al., Acc.of Chem. Res., 35:1018-25 (2002)), drug delivery (Feazell, et al., J.Am. Chem. Soc., 129(27):8438-9 (2007); Kam, et al., J. Am. Chem. Soc.,126(22):6850-1 (2004)), imaging (Sitharaman, et al., Chem. Commun.,(31):3915-7 (2005)) and biosensing (Wang and Iqbal, Journal of theMinerals, 57:27-29 (2005)).

1. Methods for Making CNTs

CNTs may be fabricated using any suitable method. CNTs are normallyproduced by various methods, such as arc-discharge methods, laserevaporation methods, thermal chemical vapor deposition (CVD) methods,and flowing vapor deposition methods. The arc-discharge method is amethod of growing CNTs by means of arc discharge using carbonelectrodes. The arc-discharge method is capable of producing an enormousamount of CNTs. The laser evaporation method typically forms CNTs byevaporating part of a graphite electrode by means of a laser. Thethermal CVD method grows carbon nanotubes at a high temperature bythermally decomposing hydrocarbon, which is a carbon source, on asubstrate with a metal catalyst thereon. The flowing vapor depositionmethod generates carbon nanotubes by making an organic transition metalcompound and a hydrocarbon compound, which is a carbon source, bothflowing with a carrier gas, react with each other at a high temperature.

2. Methods for Attaching Proteins to CNTs

The CNT compositions contain attached proteins. Proteins may be attachedto CNTs covalently through reaction with the functionalized CNT surfaceor non-covalently by non-specific adsorption (Kam, et al., J. Am. Chem.Soc., 126(22):6850-1 (2004); Karajanagi, et al., Langmuir, 20:11594-9(2004)).

CNTs have a high capacity for protein adsorption due to their highsurface area. The surface area of CNTs available for protein adsorptionmay also be adjusted by altering the surface chemistry of the CNT. Inthis way, accessible surfaces that are a priori not available forprotein adsorption may be made accessible through chemical treatment. Inone embodiment, CNTs are subjected to treatment with acid prior toprotein adsorption. Recent studies have demonstrated that acid treatmentof SWNTs induces defects on the surface of the nanotubes (Hu, et al.,Jour. Phys. Chem. B, 107:13838-42 (2003)), as well as promotede-bundling (Liang, et al., Nano Lett., 4:1257-60 (2004)), which can becorrelated with an increase in surface area (Hemraj-Benny, et al., Jour.Coll. Interf. Sci., 317(2):375-82 (2008)). In one embodiment, CNTs aretreated with nitric acid prior to protein adsorption, which introducescarboxylic acid groups at the open ends leading to sites of defects andhence increasing the capacity for protein adsorption (Hu, et al., Jour.Phys. Chem. B, 107:13838-42 (2003)). In one embodiment, the CNTs arereduced following acid treatment. For example, following nitric acidtreatment, CNTs may be treated with lithium borohydride topreferentially reduce the oxygenated groups created by the acidtreatment, favoring the dispersion of the CNTs in solution (U.S.Published Application No. 2004/0232073) and further increasing thesurface area available for protein adsorption. The examples belowdemonstrate that treatment of CNTs with 3M HNO₃ significantly increasessurface area of SWNTs, which is further increased by subsequenttreatment with LiBH₄.

In addition to non-specific adsorption, proteins can also be attached toCNTs through covalent interactions through various functional groups.Functionality refers to conjugation of a molecule to the surface of theCNT via a functional chemical group (carboxylic acids, aldehydes,amines, sulfhydryls and hydroxyls) present on the CNT and present on themolecule to be attached. Biochemical functionalization of CNTs usingvarious proteins for potential applications in biological systems aredescribed by Kam, et al., J. Am. Chem. Soc., 126(22):6850-1 (2004);Bianco, et al., Curr. Opin. Chem. Biol., 9(6):674-9 (2005); Pantarotto,et al., J. Am. Chem. Soc., 125(20):6160-4 (2003); Williams, et al.,Nature, 420(6917):761 (2002); Pamtarotto, et al., Chem. Commun., 1:16-7(2004).

B. CNT-Based Artificial Antigen Presenting Cells (aAPCs)

In one embodiment, the CNT compositions function as aAPCs. In thisembodiment, proteins that are covalently or non-covalently attached toCNTs are T cell ligands that bind to cell surface molecules on T cells.Typically, the ligands are polypeptides. Suitable T cell ligandsinclude, but are not limited to, antigen-specific and polyclonal T cellreceptor ligands, co-stimulatory molecules, and T cell targeting andadhesion molecules. CNT aAPCs may be associated with a single species offunctional T cell ligand or may be associated with any combination ofdisclosed T cell ligands in any ratio.

Suitable T cell ligands may contain the entire protein that binds to thedesired cell surface receptor, or may contain only a portion of theligand. For example, it may be desirable to remove a portion of theligand that has an undesirable biological activity, or it may bedesirable to remove a portion of the ligand to enable attachment of theCNT. The only requirement when a portion of a ligand is present is thatthe portion of the ligand substantially retains the ligand's receptorbinding activity. The terms “portion” and “fragment” are used hereininterchangeably.

Suitable T cell ligands include variant ligands. As used herein, a“variant” polypeptide contains at least one amino acid sequencealteration as compared to the amino acid sequence of the correspondingwild-type polypeptide. An amino acid sequence alteration can be, forexample, a substitution, a deletion, or an insertion of one or moreamino acids.

A variant polypeptide can have any combination of amino acidsubstitutions, deletions or insertions. In one embodiment, variantpolypeptides have an integer number of amino acid alterations such thattheir amino acid sequence shares at least 60, 70, 80, 85, 90, 95, 97,98, 99, 99.5 or 100% identity with an amino acid sequence of acorresponding wild type amino acid sequence. In a preferred embodiment,variant polypeptides have an amino acid sequence sharing at least 60,70, 80, 85, 90, 95, 97, 98, 99, 99.5 or 100% identity with the aminoacid sequence of a corresponding wild type polypeptide.

Percent sequence identity can be calculated using computer programs ordirect sequence comparison. Preferred computer program methods todetermine identity between two sequences include, but are not limitedto, the GCG program package, FASTA, BLASTP, and TBLASTN (see, e.g., D.W. Mount, 2001, Bioinformatics: Sequence and Genome Analysis, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The BLASTPand TBLASTN programs are publicly available from NCBI and other sources.The well-known Smith Waterman algorithm may also be used to determineidentity.

Exemplary parameters for amino acid sequence comparison include thefollowing: 1) algorithm from Needleman and Wunsch (J. Mol. Biol.,48:443-453 (1970)); 2) BLOSSUM62 comparison matrix from Hentikoff andHentikoff (Proc. Natl. Acad. Sci. U.S.A., 89:10915-10919 (1992)) 3) gappenalty=12; and 4) gap length penalty=4. A program useful with theseparameters is publicly available as the “gap” program (Genetics ComputerGroup, Madison, Wis.). The aforementioned parameters are the defaultparameters for polypeptide comparisons (with no penalty for end gaps).

Alternatively, polypeptide sequence identity can be calculated using thefollowing equation: % identity=(the number of identicalresidues)/(alignment length in amino acid residues)*100. For thiscalculation, alignment length includes internal gaps but does notinclude terminal gaps.

Amino acid substitutions in variant polypeptides may be “conservative”or “non-conservative”. As used herein, “conservative” amino acidsubstitutions are substitutions wherein the substituted amino acid hassimilar structural or chemical properties, and “non-conservative” aminoacid substitutions are those in which the charge, hydrophobicity, orbulk of the substituted amino acid is significantly altered.Non-conservative substitutions will differ more significantly in theireffect on maintaining (a) the structure of the peptide backbone in thearea of the substitution, for example, as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

Examples of conservative amino acid substitutions include those in whichthe substitution is within one of the five following groups: 1) smallaliphatic, nonpolar or slightly polar residues (Ala, Ser, Thr, Pro,Gly); 2) polar, negatively charged residues and their amides (Asp, Asn,Glu, Gln); polar, positively charged residues (His, Arg, Lys); largealiphatic, nonpolar residues (Met, Leu, Ile, Val, Cys); and largearomatic resides (Phe, Tyr, Trp). Examples of non-conservative aminoacid substitutions are those where 1) a hydrophilic residue, e.g., serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.,leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; 2) a cysteine orproline is substituted for (or by) any other residue; 3) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or 4) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) a residue that does not have aside chain, e.g., glycine.

Variant polypeptides may be modified by chemical moieties that may bepresent in polypeptides in a normal cellular environment, for example,phosphorylation, methylation, amidation, sulfation, acylation,glycosylation, sumoylation and ubiquitylation. Variant polypeptides mayalso be modified with a label capable of providing a detectable signal,either directly or indirectly, including, but not limited to,radioisotopes and fluorescent compounds.

Variant polypeptides may also be modified by chemical moieties that arenot normally added to polypeptides in a cellular environment. Suchmodifications may be introduced into the molecule by reacting targetedamino acid residues of the polypeptide with an organic derivatizingagent that is capable of reacting with selected side chains or terminalresidues. Another modification is cyclization of the protein.

Examples of chemical derivatives of the polypeptides include lysinyl andamino terminal residues derivatized with succinic or other carboxylicacid anhydrides. Derivatization with a cyclic carboxylic anhydride hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate. Carboxyl side groups, aspartyl or glutamyl, may beselectively modified by reaction with carbodiimides (R—N═C═N—R′) such as1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonia. Polypeptides may alsoinclude one or more D-amino acids that are substituted for one or moreL-amino acids.

Polypeptides to be attached to CNTs may also be coupled to otherpolypeptides to form fusion proteins. Exemplary polypeptides have afirst fusion partner including all or a part of a T cell ligand fused(i) directly to a second polypeptide or, (ii) optionally, fused to alinker peptide sequence that is fused to the second polypeptide.

1. T Cell Receptor Activators

a. Antigen-Specific T Cell Activators

Antigen molecules are recognized by the immune system after internalprocessing by natural APCs (Lanzavecchia, Curr. Opin. Immunol., 8:348-54(1996)). In order to present an antigen, the antigen is broken down intosmall peptidic fragments by enzymes contained in vesicles in thecytoplasm of the APCs. The enzymes are part of a complex of proteolyticenzymes called a proteosome. Most cells have several different types ofproteosomes with differing combinations of specificities, which they useto recycle their intracellular proteins. The peptides produced by theproteosomes are generated in the cytosol and transported into the Golgi,where they are linked to cellular major histocompatibility complex (MHC)molecules. These are referred to as human leukocyte antigens, or “HLAs”,in human. MHC and HLA are used interchangeably herein unless specifiedotherwise.

i. HLA and MHC Molecules

In one embodiment, the CNT aAPCs described herein containantigen-presenting molecules having determinants which match that of aselected subject or which match any known antigen-presenting moleculedeterminants. The antigen-presenting molecules may be MHC/HLA class I orclass II molecules.

There are two types of HLA molecules used for antigen presentation,class I and class II molecules. HLA class I molecules are expressed onthe surface of all cells and HLA class II are expressed on the surfaceof a specialized class of cells called professional APCs. HLA class IImolecules bind primarily to peptides derived from proteins made outsideof an APC, but can present self (endogenous) antigens. In contrast, HLAclass I molecules bind to peptides derived from proteins made inside acell, including proteins expressed by an infectious agent (e.g., such asa virus) in the cell and by a tumor cell. When the HLA class I proteinsreach the surface of the cell these molecules will thus display any oneof many peptides derived from the cytosolic proteins of that cell, alongwith normal “self” peptides being synthesized by the cell. Peptidespresented in this way are recognized by T-cell receptors which engageT-lymphocytes in an immune response against the antigens to induceantigen-specific cellular immunity.

Class I transplantation antigens of the major histocompatibility complex(MHC) or HLA are cell surface glycoproteins which present antigens tocytotoxic T-cells. They are heterodimeric and composed of a polymorphic,MHC-encoded, approximately 45 kD heavy chain, which is non-covalentlyassociated with an approximately 12 kD β-2 microglobulin (β-2m) lightchain.

The extracellular portion of the MHC Class I heavy chain is divided intothree domains, α-1, α-2, and α-3, each approximately 90 amino acids longand encoded on separate exons. The α-3 domain and β-2m are relativelyconserved and show amino-acid sequence homology to immunoglobulinconstant domains. The polymorphic α-1 and α-2 domains show nosignificant sequence homology to immunoglobulin constant or variableregion, but do have weak sequence homology to each other. Themembrane-distal polymorphic α-1 (approximately 90 amino acids) and α-2(approximately 92 amino acids) domains each include four anti-parallel,β-pleated sheets bordered by one a-helical regions, (the first from theα-1 and the second from the α-2 domain). The α-2 domain is attached tothe less-polymorphic, membrane-proximal α-3 (approximately 92 aminoacids) domain which is followed by a conserved transmembrane (25 aminoacids) and an intra-cytoplasmic (approximately 30 amino acids) segment.The rat, mouse, and human Class I MHC molecules are believed to havesimilar structural characteristics based upon known nucleotide sequencesof the various MHC Class I molecules.

The classical class I gene family includes the highly polymorphic humanclass I molecules HLA-A, -B, and -C, and murine class I (i.e., H-2)molecules D, K, and L. A series of structural relatives (non-classicalclass I molecules) has been found in humans (e.g., HLA-E, -F, -G, -H,-I, and -J; and CD1) and mice (Q, T, M, and CD1) (Shawar, et al., Annu.Rev. Immunol., 12:839-880 (1994)). These molecules have the typicalstructure of an antigen-presenting molecule, where a polymorphic heavychain is noncovalently associated with the conserved β2-M subunit.

In the case of human class I determinants, the determinant can be apolypeptide encoded by any of the known HLA genetic loci, as well aspolypeptides encoded by genetic loci not yet discovered so long as thesecan present antigen to a T cell in a manner effective to activate the Tcell receptor. Examples of known HLA class I genetic loci include forHLA-A: A1, A2, A3, A11, A23, A24, A25, A26, A28, A29, A30, A31, A32 andAw33; for HLA-B: B7, B13, B18, B27, B35, B37, B38, B39, Bw31, Bw42, B44,B45, B49, Bw50, B51, Bw52, Bw53, Bw54, Bw55, Bw57, Bw58, Bw60, Bw61,Bw62, Bw63, Bw64 and Bw65; for HLA-C: Cw1^(b), Cw2, Cw3, Cw4, Cw5, Cw6,Cw7 and Cw8.

The amino acid sequences of mammalian MHC class II alpha and beta chainproteins, as well as nucleic acids encoding these proteins, are alsowell known in the art and available from numerous sources includingGenBank. Exemplary sequences are provided in Auffray, et al., Nature,308(5957):327-333 (1984) (human HLA DQα); Larhammar, et al., Proc. Natl.Acad. Sci. USA., 80(23):7313-7317 (1983) (human LILA DQβ); Das, et al.,Proc. Natl. Acad. Sci. USA., 80 (12): 3543-3547 (1983) (human HLA DRα);Tonnelle, et al., EMBO J., 4(11):2839-2847 (1985) (human HLA DRβ);Lawrence, et al., Nucleic Acids Res., 13(20):7515-7528 (1985) (human HLADPα); and Kelly and Trowsdale, Nucl. Acids Res., 13(5):1607-1621 (1985)(human HLA DPβ).

The MHC class I or class II polypeptide selected for use with the CNTaAPCs is typically encoded by genetic loci present in the subject to betreated.

ii. Antigens

MHC/HLA class I or class II molecules are used to present antigens to Tcells to activate and expand T cells specific to the antigen. Antigenscan be peptides, polypeptides, proteins, polysaccharides, saccharides,lipids, nucleic acids, or combinations thereof. Because CTL epitopesusually comprise 8-10 amino acid long (Townsend, et al., Annu. Rev.Immunol., 7:601-624 (1989); Monaco, Cell, 54:777-785 (1992); Yewdell, etal., Adv. in Immunol., 52:1-123 (1992)), in one embodiment, antigens areshort polypeptides. Antigenic polypeptides may be about 5 to 40 aminoacids, preferably 6 to 25 amino acids, more preferably 8 to 10 aminoacids, in length. Examples of antigens presented in various immuneresponses are described in more detail below and are generally known inthe art (Engelhard, Curr. Opin. Immun., 6:13-23 (1994)).

Suitable antigens are known in the art and are available from commercialgovernment and scientific sources. Criteria for identifying andselecting effective antigenic peptides (e.g., minimal peptide sequencescapable of eliciting an immune response) can be found in the art. Forexample, Apostolopoulos, et al. (Curr. Opin. Mol. Ther., 2:29-36(2000)), discusses the strategy for identifying minimal antigenicpeptide sequences based on an understanding of the three-dimensionalstructure of an antigen-presenting molecule and its interaction withboth an antigenic peptide and T-cell receptor. Shastri, (Curr. Opin.Immunol., 8:271-7 (1996)), disclose how to distinguish rare peptidesthat serve to activate T cells from the thousands peptides normallybound to MHC molecules.

The antigen can be derived from any source including, but not limitedto, a virus, bacterium, parasite, plant, protozoan, fungus, tissue ortransformed cell such as a cancer or leukemic cell. The antigens may bepurified or partially purified polypeptides derived from tumors or viralor bacterial sources. The antigens can be recombinant polypeptidesproduced by expressing DNA encoding the polypeptide antigen in aheterologous expression system. The antigens can be DNA encoding all orpart of an antigenic polypeptide. The DNA may be in the form of vectorDNA such as plasmid DNA.

Antigens may be provided as single antigens or may be provided incombination. Antigens may also be provided as complex mixtures ofpolypeptides or nucleic acids.

Viral Antigens

A viral antigen can be isolated from any virus including, but notlimited to, a virus from any of the following viral families:Arenaviridae, Arterivirus, Astroviridae, Baculoviridae, Badnavirus,Barnaviridae, Birnaviridae, Bromoviridae, Bunyaviridae, Caliciviridae,Capillovirus, Carlavirus, Caulimovirus, Circoviridae, Closterovirus,Comoviridae, Coronaviridae (e.g., Coronavirus, such as severe acuterespiratory syndrome (SARS) virus), Corticoviridae, Cystoviridae,Deltavirus, Dianthovirus, Enamovirus, Filoviridae (e.g., Marburg virusand Ebola virus (e.g., Zaire, Reston, Ivory Coast, or Sudan strain)),Flaviviridae, (e.g., Hepatitis C virus, Dengue virus 1, Dengue virus 2,Dengue virus 3, and Dengue virus 4), Hepadnaviridae, Herpesviridae(e.g., Human herpesvirus 1, 3, 4, 5, and 6, and Cytomegalovirus),Hypoviridae, Iridoviridae, Leviviridae, Lipothrixviridae, Microviridae,Orthomyxoviridae (e.g., Influenzavirus A and B and C), Papovaviridae,Paramyxoviridae (e.g., measles, mumps, and human respiratory syncytialvirus), Parvoviridae, Picornaviridae (e.g., poliovirus, rhinovirus,hepatovirus, and aphthovirus), Poxviridae (e.g., vaccinia and smallpoxvirus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., lentivirus,such as human immunodeficiency virus (HIV) 1 and HIV 2), Rhabdoviridae(for example, rabies virus, measles virus, respiratory syncytial virus,etc.), Togaviridae (for example, rubella virus, dengue virus, etc.), andTotiviridae. Suitable viral antigens also include all or part of Dengueprotein M, Dengue protein E, Dengue D1NS1, Dengue D1NS2, and DengueD1NS3.

Viral antigens may be derived from a particular strain such as apapilloma virus, a herpes virus, i.e. herpes simplex 1 and 2; ahepatitis virus, for example, hepatitis A virus (HAV), hepatitis B virus(HBV), hepatitis C virus (HCV), the delta hepatitis D virus (HDV),hepatitis E virus (HEV) and hepatitis G virus (HGV), the tick-borneencephalitis viruses; parainfluenza, varicella-zoster, cytomeglavirus,Epstein-Barr, rotavirus, rhinovirus, adenovirus, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, and lymphocytic choriomeningitis.

Bacterial Antigens

Bacterial antigens can originate from any bacteria including, but notlimited to, Actinomyces, Anabaena, Bacillus, Bacteroides, Bdellovibrio,Bordetella, Borrelia, Campylobacter, Caulobacter, Chlamydia, Chlorobium,Chromatium, Clostridium, Corynebacterium, Cytophaga, Deinococcus,Escherichia, Francisella, Halobacterium, Heliobacter, Haemophilus,Hemophilus influenza type B (HIB), Hyphomicrobium, Legionella,Leptspirosis, Listeria, Meningococcus A, B and C, Methanobacterium,Micrococcus, Myobacterium, Mycoplasma, Myxococcus, Neisseria,Nitrobacter, Oscillatoria, Prochloron, Proteus, Pseudomonas,Phodospirillum, Rickettsia, Salmonella, Shigella, Spirillum,Spirochaeta, Staphylococcus, Streptococcus, Streptomyces, Sulfolobus,Thermoplasma, Thiobacillus, and Treponema, Vibrio, and Yersinia.

Parasite Antigens

Parasite antigens can be obtained from parasites such as, but notlimited to, an antigen derived from Cryptococcus neoformans, Histoplasmacapsulatum, Candida albicans, Candida tropicalis, Nocardia asteroides,Rickettsia ricketsii, Rickettsia typhi, Mycoplasma pneumoniae,Chlamydial psittaci, Chlamydial trachomatis, Plasmodium falciparum,Trypanosoma brucei, Entamoeba histolytica, Toxoplasma gondii,Trichomonas vaginalis and Schistosoma mansoni. These include Sporozoanantigens, Plasmodian antigens, such as all or part of a Circumsporozoiteprotein, a Sporozoite surface protein, a liver stage antigen, an apicalmembrane associated protein, or a Merozoite surface protein.

Allergens and Environmental Antigens

The antigen can be an allergen or environmental antigen, such as, butnot limited to, an antigen derived from naturally occurring allergenssuch as pollen allergens (tree-, herb, weed-, and grass pollenallergens), insect allergens (inhalant, saliva and venom allergens),animal hair and dandruff allergens, and food allergens. Important pollenallergens from trees, grasses and herbs originate from the taxonomicorders of Fagales, Oleales, Pinales and platanaceae including i.e. birch(Betula), alder (Alnus), hazel (Corylus), hornbeam (Carpinus) and olive(Olea), cedar (Cryptomeriaand Jumperus), Plane tree (Platanus), theorder of Poales including i.e. grasses of the genera Lolium, Phleum,Poa, Cynodon, Dactylis, Holcus, Phalaris, Secale, and Sorghum, theorders of Asterales and Urticales including i.a. herbs of the generaAmbrosia, Artemisia, and Parietaria. Other allergen antigens that may beused include allergens from house dust mites of the genusDermatophagoides and Euroglyphus, storage mite e.g. Lepidoglyphys,Glycyphagus and Tyrophagus, those from cockroaches, midges and flease.g. Blatella, Periplaneta, Chironomus and Ctenocepphalides, those frommammals such as cat, dog and horse, birds, venom allergens includingsuch originating from stinging or biting insects such as those from thetaxonomic order of Hymenoptera including bees (superfamily Apidae),wasps (superfamily Vespidea), and ants (superfamily Formicoidae). Stillother allergen antigens that may be used include inhalation allergensfrom fungi such as from the genera Alternaria and Cladosporium.

Tumor Antigens

The antigen can be a tumor antigen, including a tumor-associated ortumor-specific antigen, such as, but not limited to, alpha-actinin-4,Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a,coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein,LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2,KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9,pm1-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras,Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-me1, Lage-1, Mage-A1,2,3,4,6,10,12,Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA(MART-I), gp100 (Pme1 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3,BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1,Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET,IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, humanpapillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5,MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9,CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA,PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG,BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50,CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344,MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP,and TPS.

Self Antigens or Autoantigens

The antigen may also be a self-antigen or an autoantigen. Antigens maybe antigens of any autoimmune or inflammatory disease or disorderincluding, but not limited to, diabetes mellitus, arthritis (includingrheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis,psoriatic arthritis), multiple sclerosis, myasthenia gravis, systemiclupus erythematosis, autoimmune thyroiditis, dermatitis (includingatopic dermatitis and eczematous dermatitis), psoriasis, Sjogren'sSyndrome, including keratoconjunctivitis sicca secondary to Sjogren'sSyndrome, alopecia greata, allergic responses due to arthropod bitereactions, Crohn's disease, ulcer, iritis, conjunctivitis,keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drugeruptions, leprosy reversal reactions, erythema nodosum leprosum,autoimmune uveitis, allergic encephalomyelitis, acute necrotizinghemorrhagic encephalopathy, idiopathic bilateral progressivesensorineural hearing loss, aplastic anemia, pure red cell anemia,idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis,chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue,lichen planus, Crohn's disease, Graves ophthalmopathy, sarcoidosis,primary biliary cirrhosis, uveitis posterior, and interstitial lungfibrosis.

Preferred autoantigens include, but are not limited to, at least aportion of a thyroid-stimulating hormone receptor, pancreatic P cellantigens, epidermal cadherin, acetyl choline receptor, plateletantigens, nucleic acids, nucleic acid protein complexes, myelin protein,thyroid antigens, joint antigens, antigens of the nervous system,salivary gland proteins, skin antigens, kidney antigens, heart antigens,lung antigens, eye antigens, erythrocyte antigens, liver antigens andstomach antigens.

Examples of antigens involved in autoimmune disease include glutamicacid decarboxylase 65 (GAD 65), native DNA, myelin basic protein, myelinproteolipid protein, acetylcholine receptor components, thyroglobulin,and the thyroid stimulating hormone (TSH) receptor.

Examples of antigens involved in graft rejection include antigeniccomponents of the graft to be transplanted into the graft recipient suchas heart, lung, liver, pancreas, kidney, and neural graft components.

b. Polyclonal T Cell Activators

In another embodiment, the CNT aAPCs contain polyclonal T cell receptoractivators that activate T cells in the absence of specific antigens.Suitable polyclonal T cell activators include the mitogenic lectinsconcanavalin-A (ConA) phytohemagglutinin (PHA) and pokeweed mitogen(PWM).

Other suitable polyclonal T cell activators include antibodies thatcrosslink the T cell receptor/CD3 complex. Exemplary antibodies thatcrosslink the T cell receptor include the HIT3a, UCHT1 and OKT3monoclonal antibodies.

2. Costimulatory and T Cell Adhesion Molecules

In addition to ligation of the T cell receptor, activation andproliferation of lymphocytes are regulated by both positive and negativesignals from costimulatory molecules. The most extensively characterizedT cell costimulatory pathway is B7-CD28, in which B7-1 (CD80) and B7-2(CD86) each can engage the stimulatory CD28 receptor and the inhibitoryCTLA-4 (CD152) receptor. In conjunction with signaling through the Tcell receptor, CD28 ligation increases antigen-specific proliferation ofT cells, enhances production of cytokines, stimulates differentiationand effector function, and promotes survival of T cells (Lenshow, etal., Annu. Rev. Immunol., 14:233-258 (1996); Chambers and Allison, Curr.Opin. Immunol., 9:396-404 (1997); and Rathmell and Thompson, Annu. Rev.Immunol., 17:781-828 (1999)).

The CNT aAPCs described herein may contain one or more species ofco-stimulatory molecule. Exemplary co-stimulatory molecules, alsoreferred to as “co-stimulators”, include, but are not limited to, CD7,B7-1 (CD80), B7-2 (CD86), PD-L1, PD-L2, 4-1BBL, OX40L, inducibleco-stimulatory ligand (ICOS-L), intercellular adhesion molecule (ICAM),CD2, CD5, CD9, CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM,lymphotoxin beta receptor, 3/TR6, ILT3, ILT4, HVEM, an agonist orantibody that binds Toll ligand receptor and a ligand that specificallybinds with B7-H3. Other exemplary co-stimulatory molecules that can beused include antibodies that specifically bind with a co-stimulatorymolecule present on a T cell, such as, but not limited to, CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associatedantigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand thatspecifically binds with CD83. Other suitable costimulatory moleculesinclude, but are not limited to, costimulatory variants and fragments ofthe natural ligands described above.

Adhesion molecules may be included for the purpose of enhancing thebinding association between the CNT aAPCs and T cells. Suitable adhesionmolecules include, but are not limited to, LFA-1, CD49d/29(VLA-4),CD11a/18, CD54(ICAM-1), and CD106(VCAM) and antibodies to their ligands.Other suitable adhesion molecules include antibodies to selectins L, E,and P.

C. Composite Nanotube Particulate Compositions

In one embodiment, CNT compositions that function as aAPCs are compositecarbon nanotube-polymer nanoparticle (CNP) compositions. In thisembodiment, proteins that are covalently or non-covalently attached toCNTs are T cell ligands that bind to cell surface molecules on T cells.In some embodiments, any of the CNT aAPCs described above can be used ina CNP composition. The CNP compositions have bound to or present on theCNT surface one or more polymer nanoparticles. The nanoparticles aretypically composed of a biodegradable and biocompatible polymer matrix.Immunostimulatory agents are present on, incorporated within, orassociated covalently or non-covalently with the polymer matrix. Theexamples presented below demonstrate that CNP compositions enhanced Tcell stimulation to a level comparable to clinically relevant standardsusing a thousand-fold less soluble IL-2. CNP-expanded T cells showenhanced effector function and significantly delay tumor growth. Theseresults demonstrate the potential of CNP compositions for T cellimmunotherapy, especially for adoptive immunotherapies.

In preferred embodiments the CNP compositions containing a T cell ligandand a nanoparticle contain one or more magnetic particles. The magneticparticles will in some cases be present on or encapsulated within thenanoparticle. In some embodiments the magnetic particles will be asecond nanoparticle that is bound to or present on the CNT surface, orwill be present on or encapsulated within a second nanoparticle that isbound to or present on the CNT surface. The examples presented belowdemonstrate that CNPs containing magnetic particles exhibited roomtemperature superparamagnetic properties. Room temperature magnetismallowed for magnetic separation of activated T cells after CNPenrichment.

1. Antigens

The disclosed CNPs may contain any suitable antigen or combination ofantigens. Exemplary antigens are discussed above with respect to CNTaAPC compositions.

2. Polymer Nanoparticles

The CNP compositions have bound to or present on the CNT surface one ormore polymer nanoparticles. In some embodiments, a therapeutic,diagnostic, and/or prophylactic agent is covalently associated with apolymeric matrix. An immunostimulatory agent can in associated with thepolymeric matrix or encapsulated within the polymeric matrix.Association can be covalent or non-covalent. In some embodiments,covalent association is mediated by a linker (e.g., an aliphatic orheteroaliphatic linker). In some embodiments, a therapeutic, diagnostic,and/or prophylactic agent is non-covalently associated with a polymericmatrix. In some embodiments, a therapeutic, diagnostic, and/orprophylactic agent is associated with the surface of, encapsulatedwithin, surrounded by, and/or dispersed throughout a polymeric matrix.

A wide variety of polymers and methods for forming particles therefromare known in the art of drug delivery. In some embodiments, the matrixof a particle comprises one or more polymers. Polymers may be natural orunnatural (synthetic) polymers. Polymers may be homopolymers orcopolymers comprising two or more monomers. In terms of sequence,copolymers may be random, block, or comprise a combination of random andblock sequences. Typically, polymers are organic polymers.

Examples of polymers include polyalkylenes (e.g., polyethylenes),polycarbonates (e.g., poly(1,3-dioxan-2one)), polyanhydrides (e.g.,poly(sebacic anhydride)), polyhydroxyacids (e.g.,poly(beta.-hydroxyalkanoate)), polyfumarates, polycaprolactones,polyamides (e.g., polycaprolactam), polyacetals, polyethers, polyesters(e.g., polylactide, polyglycolide), poly(orthoesters), polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, poly(arylates), polycarbonates, poly(propylene fumarates),polyhydroxyalkanoates, polyketals, polyesteramides, poly(dioxanones),polyhydroxybutyrates, polyhydroxyvalyrates, polyorthocarbonates,poly(vinyl pyrrolidone), polyalkylene oxalates, polyalkylene succinates,poly(malic acid), poly(methyl vinyl ether), and poly(maleic anhydride).In some embodiments, polymers include polymers which have been approvedfor use in humans by the United States Food and Drug Administration(U.S.F.D.A.) under 21 C.F.R. .sctn.177.2600, including but not limitedto polyesters (e.g., polylactic acid, polyglycolic acid,poly(lactic-co-glycolic acid)), polycaprolactone, polyvalerolactone,poly(1,3-dioxan-2one)); polyanhydrides (e.g., poly(sebacic anhydride));polyethers (e.g., polyethylene glycol); polyurethanes;polymethacrylates; polyacrylates; and polycyanoacrylates.

In some embodiments, polymers can be hydrophilic. For example, polymersmay comprise anionic groups (e.g., phosphate group, sulphate group,carboxylate group); cationic groups (e.g., quaternary amine group); orpolar groups (e.g., hydroxyl group, thiol group, amine group).

In some embodiments, polymers may be modified with one or more moietiesand/or functional groups. Any moiety or functional group can be used. Insome embodiments, polymers may be modified with polyethylene glycol(PEG), with a carbohydrate, and/or with acyclic polyacetals derived frompolysaccharides (Papisov, 2001, ACS Symposium Series, 786:301). In someembodiments, polymers may be modified with PEG.

In some embodiments, polymers may be modified with a lipid or fatty acidgroup, properties of which are described in further detail below. Insome embodiments, a fatty acid group may be one or more of butyric,caproic, caprylic, capric, lauric, myristic, palmitic, stearic,arachidic, behenic, or lignoceric acid. In some embodiments, a fattyacid group may be one or more of palmitoleic, oleic, vaccenic, linoleic,alpha-linoleic, gamma-linoleic, arachidonic, gadoleic, arachidonic,eicosapentaenoic, docosahexaenoic, or erucic acid.

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such as poly(lacticacid-co-glycolic acid) and poly(lactide-co-glycolide), collectivelyreferred to herein as “PLGA”; and homopolymers comprising glycolic acidunits, referred to herein as “PGA,” and lactic acid units, such aspoly-L-lactic acid, poly-D-lactic acid, poly-D,L-lactic acid,poly-L-lactide, poly-D-lactide, and poly-D,L-lactide, collectivelyreferred to herein as “PLA.” In some embodiments, exemplary polyestersinclude, for example, polyhydroxyacids; lactide-PEG copolymers (e.g.,PLA-PEG copolymers); glycolide-PEG copolymers (e.g., PGA-PEGcopolymers); copolymers of lactide and glycolide (e.g., PLGA);copolymers of lactide, glycolide, and PEG (e.g., PLGA-PEG copolymers);and derivatives thereof In some embodiments, polyesters include, forexample, polyanhydrides, poly(ortho ester), poly(ortho ester)-PEGcopolymers, poly(caprolactone), poly(caprolactone)-PEG copolymers,polylysine, polylysine-PEG copolymers, poly(ethylene imine),poly(ethylene imine)-PEG copolymers, poly(L-lactide-co-L-lysine),poly(serine ester), poly(4-hydroxy-L-proline ester),poly[.alpha.-(4-aminobutyl)-L-glycolic acid], and derivatives thereof.

In certain embodiments, a polymer may be PLA. In certain embodiments, apolymer may be PGA. In certain embodiments, a polymer may be PLGA. Incertain embodiments, a polymer may be PEG. In certain embodiments, apolymer may be PEG-PLA. In certain embodiments, a polymer may bePEG-PGA. In certain embodiments, a polymer may be PEG-PLGA. In certainembodiments, a polymer may be a PEG-PLA/PLA blend. In certainembodiments, a polymer may be a PEG-PGA/PGA blend. In certainembodiments, a polymer may be a PEG-PLGA/PEG-PLGA blend. In certainembodiments, a polymer may be a PEG-PLA/PGA blend. In certainembodiments, a polymer may be PEG-PLA/PLGA blend. In certainembodiments, a polymer may be a PEG-PGA/PLA blend. In certainembodiments, a polymer may be a PEG-PLGA/PLA blend. In certainembodiments, a polymer may be a PEG-PLGA/PGA blend. In certainembodiments, a polymer may be a PEG-PGA/PLGA blend. In certainembodiments, any of the foregoing may comprise a modified PEG (e.g.methoxy(polyethylene glycol)). For example, a polymer may bemethoxy(polyethylene glycol)-PLA. In some embodiments, a polymer maycomprise any combination or blend of the foregoing.

In some embodiments, a polymer may be PLGA. PLGA is a biocompatible andbiodegradable co-polymer of lactic acid and glycolic acid, and variousforms of PLGA are characterized by the ratio of lactic acid:glycolicacid. Lactic acid can be L-lactic acid, D-lactic acid, or D,L-lacticacid. The degradation rate of PLGA can be adjusted by altering thelactic acid:glycolic acid ratio. In some embodiments, PLGA to be used ischaracterized by a lactic acid:glycolic acid ratio of approximately85:15, approximately 75:25, approximately 60:40, approximately 65:35,approximately 50:50, approximately 40:60, approximately 25:75, orapproximately 15:85.

In some embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, aminoalkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid anhydride), methyl methacrylate, polymethacrylate,poly(methyl methacrylate) copolymer, polyacrylamide, aminoalkylmethacrylate copolymer, glycidyl methacrylate copolymers,polycyanoacrylates, and combinations comprising one or more of theforegoing polymers. The acrylic polymer may comprise fully-polymerizedcopolymers of acrylic and methacrylic acid esters with a low content ofquaternary ammonium groups.

In some embodiments, polymers can be cationic polymers. In general,cationic polymers are able to condense and/or protect negatively chargedstrands of nucleic acids (e.g., DNA, RNA, or derivatives thereof).Amine-containing polymers such as poly(lysine) (Zauner et al., 1998,Adv. Drug Del. Rev., 30:97; and Kabanov et al., 1995, BioconjugateChem., 6:7), poly(ethylene imine) (PEI; Boussif et al., 1995, Proc.Natl. Acad. Sci., USA, 1995, 92:7297), and poly(amidoamine) dendrimers(Kukowska-Latallo et al., 1996, Proc. Natl. Acad. Sci., USA, 93:4897;Tang et al., 1996, Bioconjugate Chem., 7:703; and Haensler et al., 1993,Bioconjugate Chem., 4:372) are positively-charged at physiological pH,form ion pairs with nucleic acids, and mediate transfection in a varietyof cell lines.

In some embodiments, polymers can be degradable polyesters bearingcationic side chains (Putnam et al., 1999, Macromolecules, 32:3658;Barrera et al., 1993, J. Am. Chem. Soc., 115:11010; Kwon et al., 1989,Macromolecules, 22:3250; Lim et al., 1999, J. Am. Chem. Soc., 121:5633;and Zhou et al., 1990, Macromolecules, 23:3399). Examples of thesepolyesters include poly(L-lactide-co-L-lysine) (Barrera et al., 1993, J.Am. Chem. Soc., 115:11010), poly(serine ester) (Zhou et al., 1990,Macromolecules, 23:3399), poly(4-hydroxy-L-proline ester) (Putnam etal., 1999, Macromolecules, 32:3658; and Lim et al., 1999, J. Am. Chem.Soc., 121:5633). Poly(4-hydroxy-L-proline ester) was recentlydemonstrated to condense plasmid DNA through electrostatic interactions,and to mediate gene transfer (Putnam et al., 1999, Macromolecules,32:3658; and Lim et al., 1999, J. Am. Chem. Soc., 121:5633). These newpolymers are less toxic than poly(lysine) and PEI, and they degrade intonon-toxic metabolites.

In some embodiments, polymers can be anionic polymers. In someembodiments, anionic polymers comprise carboxyl, sulfate, or groups. Togive but a few examples, anionic polymers include, but are not limitedto, dextran sulfate, heparan sulfate, alginic acid, polyvinylcarboxylicacid, and arabic acid carboxymethylcellulose. In some embodiments,anionic polymers are provided as a salt (e.g., sodium salt).

In some embodiments, a polymer may be a carbohydrate, properties ofwhich are described in further detail below. In some embodiments, acarbohydrate may be a polysaccharide comprising simple sugars (or theirderivatives) connected by glycosidic bonds, as known in the art. In someembodiments, a carbohydrate may be one or more of pullulan, cellulose,microcrystalline cellulose, hydroxypropyl methylcellulose,hydroxycellulose, methylcellulose, dextran, cyclodextran, glycogen,starch, hydroxyethylstarch, carageenan, glycon, amylose, chitosan,N,O-carboxylmethylchitosan, algin and alginic acid, starch, chitin,heparin, konjac, glucommannan, pustulan, heparin, hyaluronic acid,curdlan, and xanthan.

In some embodiments, a polymer may be a protein or peptide, propertiesof which are described in further detail below. Exemplary proteinsinclude, but are not limited to, albumin, collagen, gelatin, poly(aminoacid) (e.g., polylysine), and antibodies.

In some embodiments, a polymer may be a polynucleotide. Exemplarypolynucleotides include, but are not limited to, DNA, RNA, etc.

The properties of these and other polymers and methods for preparingthem are well known in the art (see, for example, U.S. Pat. Nos.6,123,727; 5,804,178; 5,770,417; 5,736,372; 5,716,404; 6,095,148;5,837,752; 5,902,599; 5,696,175; 5,514,378; 5,512,600; 5,399,665;5,019,379; 5,010,167; 4,806,621; 4,638,045; and U.S. Pat. No. 4,946,929;Wang et al., 2001, J. Am. Chem. Soc., 123:9480; Lim et al., 2001, J. Am.Chem. Soc., 123:2460; Langer, 2000, Acc. Chem. Res., 33:94; Langer,1999, J. Control. Release, 62:7; and Uhrich et al., 1999, Chem. Rev.,99:3181). More generally, a variety of methods for synthesizing suitablepolymers are described in Concise Encyclopedia of Polymer Science andPolymeric Amines and Ammonium Salts, Ed. by Goethals, Pergamon Press,1980; Principles of Polymerization by Odian, John Wiley & Sons, FourthEdition, 2004; Contemporary Polymer Chemistry by Allcock et al.,Prentice-Hall, 1981; Deming et al., 1997, Nature, 390:386; and in U.S.Pat. Nos. 6,506,577, 6,632,922, 6,686,446, and 6,818,732.

In some embodiments, polymers can be linear or branched polymers. Insome embodiments, polymers can be dendrimers. In some embodiments,polymers can be substantially cross-linked to one another. In someembodiments, polymers can be substantially free of cross-links. In someembodiments, polymers can be used without undergoing a cross-linkingstep.

It is further to be understood that controlled release polymer systemsmay be a homopolymer, block copolymer, diblock triblock, multibockcopolymer, linear polymer, dendritic polymer, branched polymer, graftcopolymer, blend, mixture, and/or adduct of any of the foregoing andother polymers.

3. Immunostimulatory Agents

Either specific and non-specific immunostimulants can be bound to, orencapsuled within, the formulations. Suitable immunostimulants are knownand available. Suitable adjuvants are discussed below.

4. Magnetic Particles

“Magnetic material” as used herein refers to any material that induces aforce or movement when introduced into a magnetic field. Suitablemagnetic materials include, but are not limited to, ferromagnetica andsuperparamagnetic materials, such as iron containing compounds,martensitic stainless steels (e.g. 400 series), iron oxides (Fe₂O₃,Fe₃O₄), neodymium iron boron, alnico (AlNiCo), and samarium cobalt(SmCo₅).

D. CNT-Based Modular Vaccine Compositions

In another embodiment, the CNT compositions are modular vaccinecompositions that function to deliver antigens to antigen-presentingcells in vivo. In this embodiment, the CNT compositions do not directlyactivate T cells by binding to T cell surface receptors, but ratherfacilitate the delivery of large amounts of antigen to natural APCs,which, in turn, activate T cells and other immune cells. The examplesbelow demonstrate that immunization of mice with a model antigen(ovalbumin) adsorbed onto SWNTs causes priming of T cell activation to agreater extent than immunization with ovalbumin adsorbed onto alum,which is widely used as an adjuvant. Therefore, CNTs function in thisembodiment both as a carrier for antigens and also as immune adjuvants.

The CNT vaccine compositions may include elements that facilitateantigen uptake by APCs and APC activation, including, but not limitedto, additional adjuvants, dendritic cell recognition elements,epithelial cell recognition elements, or molecules which protect thecomposition from hydrolysis and degradation in low-pH environments. Thedisclosed CNT vaccine compositions are modular systems that allow forflexible addition and subtraction of these elements which allows forexquisite control over many of the variables that are important foroptimizing an effective vaccine delivery system. The modular nature ofthese vaccine compositions allows for libraries of vaccine compositionsthat may be tested for efficacy for any particular antigen.

1. Antigens

The CNT modular vaccines may contain any suitable antigen or combinationof antigens. Exemplary antigens are discussed above with respect to CNTaAPC compositions.

2. Targeting Molecules for Professional Antigen Presenting Cells

Of the main types of antigen-presenting cells (B cell, macrophages andDCs), the DC is the most potent and is responsible for initiating allantigen-specific immune responses. One biological feature of DCs istheir ability to sense conditions under which antigen is encountered,initiating a process of DC maturation. Using receptors for variousmicrobial and inflammatory products, DCs respond to antigen exposure indifferent ways depending on the nature of the pathogen (virus, bacteria,protozoan) encountered. This information is transmitted to T cells byaltered patterns of cytokine release at the time of antigen presentationin lymph nodes, altering the type of T cell response elicited. Thus,targeting DCs provides the opportunity not only to quantitativelyenhance the delivery of antigen and antigen responses in general, but toqualitatively control the nature of the immune response depending on thedesired vaccination outcome.

Dendritic cells express a number of cell surface receptors that canmediate the endocytosis of bound antigen. Targeting exogenous antigensto internalizing surface molecules on systemically-distributed antigenpresenting cells facilitates uptake of antigens and thus overcomes amajor rate-limiting step in immunization and thus in vaccination.

Dendritic cell targeting molecules include monoclonal or polyclonalantibodies or fragments thereof that recognize and bind to epitopesdisplayed on the surface of dendritic cells. Dendritic cell targetingmolecules also include ligands which bind to a cell surface receptor ondendritic cells. One such receptor, the lectin DEC-205, has been used invitro and in mice to boost both humoral (antibody-based) and cellular(CD8 T cell) responses by 2-4 orders of magnitude (Hawiger, et al., J.Exp. Med., 194(6):769-79 (2001); Bonifaz, et al., J. Exp. Med.,196(12):1627-38 (2002); Bonifaz, et al., J. Exp. Med., 199(6):815-24(2004)). In these experiments, antigens were fused to an anti-DEC205heavy chain and a recombinant antibody molecule was used forimmunization.

A variety of other endocytic receptors, including a mannose-specificlectin (mannose receptor) and IgG Fc receptors, have also been targetedin this way with similar enhancement of antigen presentation efficiency.Other suitable receptors which may be targeted include, but are notlimited to, DC-SIGN, 33D1, SIGLEC-H, DCIR, CD11c, heat shock proteinreceptors and scavenger receptors.

Other receptors which may be targeted include the toll-like receptors(TLRs). TLRs recognize and bind to pathogen-associated molecularpatterns (PAMPs). PAMPs target the TLR on the surface of the dendriticcell and signals internally, thereby potentially increasing DC antigenuptake, maturation and T-cell stimulatory capacity. PAMPs conjugated tothe particle surface or co-encapsulated include unmethylated CpG DNA(bacterial), double-stranded RNA (viral), lipopolysacharride(bacterial), peptidoglycan (bacterial), lipoarabinomannin (bacterial),zymosan (yeast), mycoplasmal lipoproteins such as MALP-2 (bacterial),flagellin (bacterial) poly(inosinic-cytidylic) acid (bacterial),lipoteichoic acid (bacterial) or imidazoquinolines (synthetic).

3. Targeting Molecules for Epithelial Cells

The potential efficacy of vaccine systems is determined in part by theirroute of administration into the body. While injection (intradermal,intramuscular, intravenous) is an acceptable solution in many cases,having a vaccine product that is orally available will greatly extendits ease of use and applicability on a global scale. For orallyadministered vaccines, epithelial cells constitute the principal barrierthat separates an organism's interior from the outside world. Epithelialcells such as those that line the gastrointestinal tract form continuousmonolayers that simultaneously confront the extracellular fluidcompartment and the extracorporeal space. Uptake of antigen by these gutepithelial cells can be enhanced, and the antigens carried by“transcytosis” to the lymphatics where they have access to dendriticcells.

In one embodiment, modular CNT vaccines may include epithelial cellrecognition elements. Epithelial cell targeting molecules includemonoclonal or polyclonal antibodies or bioactive fragments thereof thatrecognize and bind to epitopes displayed on the surface of epithelialcells. Epithelial cell targeting molecules also include ligands whichbind to a cell surface receptor on epithelial cells. Ligands include,but are not limited to, molecules such as polypeptides, nucleotides andpolysaccharides.

A variety of receptors on epithelial cells may be targeted by epithelialcell targeting molecules. Examples of suitable receptors to be targetedinclude, but are not limited to, IgE Fc receptors, EpCAM, selectedcarbohydrate specificities, dipeptidyl peptidase, and E-cadherin.

4. Additional Adjuvants

The modular CNT vaccines may include additional adjuvants. These can beincorporated into, administered with, or administered separately from,the CNT vaccine compositions.

In one embodiment the adjuvant is the synthetic glycolipidalpha-galactosylceramide (αGalCer). Dendritic cells presenting antigensin the context of CD1d can lead to rapid innate and prolonged productionof cytokines such as interferon and IL-4 by natural killer T cells (NKTcells). CD1d is a major histocompatibility complex class I-like moleculethat presents glycolipid antigens to a subset of NKT cells.Advantageously, αGalCer is not toxic to humans and has been shown to actas an adjuvant, priming both antigen-specific CD4+ and CD8+ T cellresponses. For example, it has been shown that αGalCer in conjunctionwith a malaria vaccine can lead to cytotoxic responses against infectedcells, which is an ideal scenario for vaccines against infectiousdiseases. In addition to αGalCer, other glycolipids that function asadjuvants to activate NKT cell-mediated immune responses can be used.

In another embodiment the adjuvant can be, but is not limited to, one ormore of the following: oil emulsions (e.g., Freund's adjuvant); saponinformulations; virosomes and viral-like particles; bacterial andmicrobial derivatives including, but not limited to carbohydrates suchas lipopolysachharide (LPS); immunostimulatory oligonucleotides;ADP-ribosylating toxins and detoxified derivatives; alum; BCG;mineral-containing compositions (e.g., mineral salts, such as aluminiumsalts and calcium salts, hydroxides, phosphates, sulfates, etc.);bioadhesives and/or mucoadhesives; microparticles; liposomes;polyoxyethylene ether and polyoxyethylene ester formulations;polyphosphazene; muramyl peptides; imidazoquinolone compounds; andsurface active substances (e.g. lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol).

Adjuvants may also include immunomodulators such as cytokines,interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.),interferons (e.g., interferon-.gamma.), macrophage colony stimulatingfactor, and tumor necrosis factor; and co-stimulatory molecules, such asthose of the B7 family. Such proteinaceous adjuvants may be provided asthe full-length polypeptide or an active fragment thereof, or in theform of DNA, such as plasmid DNA

5. Molecules to Inhibit Degradation of CNT Vaccine Compositions inExtreme pH Environments

CNT vaccine compositions administered orally will encounter a corrosiveenvironment in the gastrointestinal (GI) tract with areas of low andhigh pH, as well as resident degradative enzymes and solubilizingagents. For this reason, ‘shielding’ is a desired feature to protect thevaccine composition in transit to the GI epithelium. In one embodiment,modular CNT vaccine compositions further include pH-sensitive moleculeswhich protect the composition from hydrolysis and degradation in low pHenvironments. Such pH-sensitive protecting molecules are preferredbecause subsequent to the transit of the vaccine composition through alow pH environment, upon reaching its destination in the higher pHintestinal site, the CNT vaccine compositions should expose epithelialtargeting molecules to allow for specific interactions with targetepithelial cells.

Exemplary non-pH-sensitive molecules include, but are not limited to,poly(ethylene) glycol, gelatin and albumins. Exemplary pH-sensitivemolecules include, but are not limited to, elastin and poly(methacrylic)acid (PMAA). Both of these molecules are in extended conformations at pH7.4 and shrink rapidly (within seconds) upon exposure to lower pHenvironments (below pH 5 for elastin and below pH 5-6 forpoly(methacylic) acid). Other exemplary pH-sensitive molecules which maybe used include poly(acrylic acid), poly(methyl methacrylic acid) andpoly(N-alkyl acrylamides).

E. Pharmaceutically Acceptable Excipients

The CNT compositions may be administered in combination with aphysiologically or pharmaceutically acceptable carrier, excipient, orstabilizer. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients. The term“pharmaceutically-acceptable carrier” means one or more compatible solidor liquid fillers, dilutants or encapsulating substances which aresuitable for administration to a human or other vertebrate animal. Theterm “carrier” refers to an organic or inorganic ingredient, natural orsynthetic, with which the active ingredient is combined to facilitatethe application.

Pharmaceutical compositions may be formulated in a conventional mannerusing one or more physiologically acceptable carriers includingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Properformulation is dependent upon the route of administration chosen. In thepreferred embodiment, administration is by injection. Typicalformulations for injection include a carrier such as sterile saline or aphosphate buffered saline. Viscosity modifying agents and preservativesare also frequently added.

Optional pharmaceutically acceptable excipients especially for enteral,topical and mucosal administration, include, but are not limited to,diluents, binders, lubricants, disintegrants, colorants, stabilizers,and surfactants. Diluents, also referred to as “fillers,” are typicallynecessary to increase the bulk of a solid dosage form so that apractical size is provided for compression of tablets or formation ofbeads and granules. Suitable diluents include, but are not limited to,dicalcium phosphate dihydrate, calcium sulfate, lactose, sucrose,mannitol, sorbitol, cellulose, microcrystalline cellulose, kaolin,sodium chloride, dry starch, hydrolyzed starches, pregelatinized starch,silicone dioxide, titanium oxide, magnesium aluminum silicate andpowdered sugar.

Binders are used to impart cohesive qualities to a solid dosageformulation, and thus ensure that a tablet or bead or granule remainsintact after the formation of the dosage forms. Suitable bindermaterials include, but are not limited to, starch, pregelatinizedstarch, gelatin, sugars (including sucrose, glucose, dextrose, lactoseand sorbitol), polyethylene glycol, waxes, natural and synthetic gumssuch as acacia, tragacanth, sodium alginate, cellulose, includinghydroxypropylmethylcellulose, hydroxypropylcellulose, ethylcellulose,and veegum, and synthetic polymers such as acrylic acid and methacrylicacid copolymers, methacrylic acid copolymers, methyl methacrylatecopolymers, aminoalkyl methacrylate copolymers, polyacrylicacid/polymethacrylic acid and polyvinylpyrrolidone.

Lubricants are used to facilitate tablet manufacture. Examples ofsuitable lubricants include, but are not limited to, magnesium stearate,calcium stearate, stearic acid, glycerol behenate, polyethylene glycol,talc, and mineral oil.

Disintegrants are used to facilitate dosage form disintegration or“breakup” after administration, and generally include, but are notlimited to, starch, sodium starch glycolate, sodium carboxymethylstarch, sodium carboxymethylcellulose, hydroxypropyl cellulose,pregelatinized starch, clays, cellulose, alginine, gums or cross linkedpolymers, such as cross-linked PVP (POLYPLASDONE® XL from GAF ChemicalCorp).

Stabilizers are used to inhibit or retard decomposition reactions whichinclude, by way of example, oxidative reactions.

Surfactants may be anionic, cationic, amphoteric or nonionic surfaceactive agents. Suitable anionic surfactants include, but are not limitedto, those containing carboxylate, sulfonate and sulfate ions. Examplesof anionic surfactants include sodium, potassium, ammonium of long chainalkyl sulfonates and alkyl aryl sulfonates such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodium dodecylbenzenesulfonate; dialkyl sodium sulfosuccinates, such as sodiumbis-(2-ethylthioxyl)-sulfosuccinate; and alkyl sulfates such as sodiumlauryl sulfate. Cationic surfactants include, but are not limited to,quaternary ammonium compounds such as benzalkonium chloride,benzethonium chloride, cetrimonium bromide, stearyl dimethylbenzylammonium chloride, polyoxyethylene and coconut amine. Examples ofnonionic surfactants include ethylene glycol monostearate, propyleneglycol myristate, glyceryl monostearate, glyceryl stearate,polyglyceryl-4-oleate, sorbitan acylate, sucrose acylate, PEG-150laurate, PEG-400 monolaurate, polyoxyethylene monolaurate, polysorbates,polyoxyethylene octylphenylether, PEG-1000 cetyl ether, polyoxyethylenetridecyl ether, polypropylene glycol butyl ether, Poloxamer® 401,stearoyl monoisopropanolamide, and polyoxyethylene hydrogenated tallowamide. Examples of amphoteric surfactants include sodiumN-dodecyl-b-alanine, sodium N-lauryl-b-iminodipropionate,myristoamphoacetate, lauryl betaine and lauryl sulfobetaine.

If desired, the particles may also contain minor amount of nontoxicauxiliary substances such as wetting or emulsifying agents, dyes, pHbuffering agents, or preservatives.

The particles may be complexed with other agents. The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., acacia, methylcellulose, sodiumcarboxymethylcellulose, polyvinylpyrrolidone (Povidone), hydroxypropylmethylcellulose, sucrose, starch, and ethylcellulose); fillers (e.g.,corn starch, gelatin, lactose, acacia, sucrose, microcrystallinecellulose, kaolin, mannitol, dicalcium phosphate, calcium carbonate,sodium chloride, or alginic acid); lubricants (e.g. magnesium stearates,stearic acid, silicone fluid, talc, waxes, oils, and colloidal silica);and disintegrators (e.g. micro-crystalline cellulose, corn starch,sodium starch glycolate and alginic acid. If water-soluble, suchformulated complex then may be formulated in an appropriate buffer, forexample, phosphate buffered saline or other physiologically compatiblesolutions. Alternatively, if the resulting complex has poor solubilityin aqueous solvents, then it may be formulated with a non-ionicsurfactant such as TWEEN™, or polyethylene glycol. Thus, the compoundsand their physiologically acceptable solvates may be formulated foradministration.

Liquid formulations for oral administration prepared in water or otheraqueous vehicles may contain various suspending agents such asmethylcellulose, alginates, tragacanth, pectin, kelgin, carrageenan,acacia, polyvinylpyrrolidone, and polyvinyl alcohol. The liquidformulations may also include solutions, emulsions, syrups and elixirscontaining, together with the active compound(s), wetting agents,sweeteners, and coloring and flavoring agents. Various liquid and powderformulations can be prepared by conventional methods for inhalation bythe patient.

The particles may be coated. Suitable coating materials include, but arenot limited to, cellulose polymers such as cellulose acetate phthalate,hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxypropylmethylcellulose phthalate and hydroxypropyl methylcellulose acetatesuccinate; polyvinyl acetate phthalate, acrylic acid polymers andcopolymers, and methacrylic resins that are commercially available underthe trade name EUDRAGIT® (Röhm Pharma, Darmstadt, Germany), zein,shellac, and polysaccharides. Additionally, the coating material maycontain conventional carriers such as plasticizers, pigments, colorants,glidants, stabilization agents, pore formers and surfactants.

III. Methods of Use

A. T Cell Activation

The CNT aAPC and CNP compositions are useful for activating T cellseither in vivo, for active immunotherapy applications, or ex vivo, foradoptive immunotherapy applications. The CNT vaccine compositions areuseful for in vivo administration of antigens to professional APCs toelicit T cell activation for prophylactic or therapeutic applications.

The examples below demonstrate that anti-CD3 antibodies adsorbed ontoSWNT bundles stimulate cells more efficiently than equivalentconcentrations of soluble anti-CD3 antibodies. It is believed that theenhanced activity of CNT aAPCs is due to the large surface area of theCNTs and their unique aspect ratio, as well as their efficientadsorption of a large number of proteins. Furthermore, SWNT bundlesbound to anti-CD3 antibodies are more efficient at activating T cellsthan other high surface area compositions, such as activated carbon andpolystyrene and C60 nanoparticles, even when normalized by surface area.This indicates that SWNTs possess unique properties in addition to theirhigh surface area that make them ideally suited to function as scaffoldsfor aAPCs.

Activation of T cells increases their proliferation, cytokineproduction, differentiation, effector functions and/or survival. Methodsfor measuring these are well known to those in the art. The T cellsactivated by the CNT compositions can be any cell which express the Tcell receptor, including α/β and γ/δ T cell receptors. T-cells includeall cells which express CD3, including T-cell subsets which also expressCD4 and CD8. T-cells include both naive and memory cells and effectorcells such as CTL. T-cells also include regulatory cells such as Th1,Tc1, Th2, Tc2, Th3, Treg, and Trl cells. T-cells also include NKT-cellsand similar unique classes of the T-cell lineage. In preferredembodiments the T cells that are activated are CD8⁺ T cells.

1. Subjects to be Treated

In general, the CNT compositions are useful for treating a subjecthaving or being predisposed to any disease or disorder to which thesubject's immune system mounts an immune response. Treating a disease ordisorder to which the subject's immune system mounts an immune responsemay include inhibiting or delaying the development of the disease ordisorder or inhibiting or reducing the symptoms of the disease ordisorder. The compositions are useful as prophylactic compositions,which confer resistance in a subject to subsequent tumor development orexposure to infectious agents. The compositions are also useful astherapeutic compositions, which can be used to initiate or enhance asubject's immune response to a pre-existing antigen, such as a tumorantigen in a subject with cancer, or a viral antigen in a subjectinfected with a virus.

The compositions are also useful to treat or prevent diseases anddisorders characterized by undesirable activation, overactivation orinappropriate activation of the immune system, such as occurs duringallergic responses, autoimmune diseases and disorders, graft rejectionand graft-versus-host-disease. Methods for using CNT aAPCs and CNPs fortreatment of these conditions are described in more detail below.

The desired outcome of a prophylactic, therapeutic or de-sensitizedimmune response may vary according to the disease, according toprinciples well known in the art. For example, an immune responseagainst an infectious agent may completely prevent colonization andreplication of an infectious agent, affecting “sterile immunity” and theabsence of any disease symptoms. However, treatment against infectiousagents with the CNT compositions may be considered effective if itreduces the number, severity or duration of symptoms; if it reduces thenumber of individuals in a population with symptoms; or reduces thetransmission of an infectious agent. Similarly, immune responses againstcancer, allergens or infectious agents may completely treat a disease,may alleviate symptoms, or may be one facet in an overall therapeuticintervention against a disease. For example, the stimulation of animmune response against a cancer may be coupled with surgical,chemotherapeutic, radiologic, hormonal and other immunologic approachesin order to affect treatment.

a. Subjects Infected with or Exposed to Infectious Agents

In some instances, the subject can be treated prophylactically, such aswhen there may be a risk of developing disease from an infectious agent.Infectious agents include bacteria, viruses and parasites. An individualtraveling to or living in an area of endemic infectious disease may beconsidered to be at risk and a candidate for prophylactic vaccinationagainst the particular infectious agent. Preventative treatment can beapplied to any number of diseases where there is a known relationshipbetween the particular disease and a particular risk factor, such asgeographical location or work environment.

b. Subjects with or a Risk of Developing Malignant Tumors

In a mature animal, a balance usually is maintained between cell renewaland cell death in most organs and tissues. The various types of maturecells in the body have a given life span; as these cells die, new cellsare generated by the proliferation and differentiation of various typesof stem cells. Under normal circumstances, the production of new cellsis so regulated that the numbers of any particular type of cell remainconstant. Occasionally, though, cells arise that are no longerresponsive to normal growth-control mechanisms. These cells give rise toclones of cells that can expand to a considerable size, producing atumor or neoplasm. A tumor that is not capable of indefinite growth anddoes not invade the healthy surrounding tissue extensively is benign. Atumor that continues to grow and becomes progressively invasive ismalignant. The term cancer refers specifically to a malignant tumor. Inaddition to uncontrolled growth, malignant tumors exhibit metastasis. Inthis process, small clusters of cancerous cells dislodge from a tumor,invade the blood or lymphatic vessels, and are carried to other tissues,where they continue to proliferate. In this way a primary tumor at onesite can give rise to a secondary tumor at another site. Thecompositions and method described herein may be useful for treatingsubjects having malignant tumors. Treating a subject having a malignanttumor includes delaying or inhibiting the growth of a tumor in asubject, reducing the growth or size of the tumor, inhibiting orreducing metastasis of the tumor, and inhibiting or reducing symptomsassociated with tumor development or growth.

Malignant tumors which may be treated are classified herein according tothe embryonic origin of the tissue from which the tumor is derived.Carcinomas are tumors arising from endodermal or ectodermal tissues suchas skin or the epithelial lining of internal organs and glands. Amelanoma is a type of carcinoma of the skin for which this technology isparticularly useful. Sarcomas, which arise less frequently, are derivedfrom mesodermal connective tissues such as bone, fat, and cartilage. Theleukemias and lymphomas are malignant tumors of hematopoietic cells ofthe bone marrow. Leukemias proliferate as single cells, whereaslymphomas tend to grow as tumor masses. Malignant tumors may show up atnumerous organs or tissues of the body to establish a cancer.

The types of cancer that can be treated in with the providedcompositions and methods include, but are not limited to, the following:bladder, brain, breast, cervical, colo-rectal, esophageal, kidney,liver, lung, nasopharangeal, pancreatic, prostate, skin, stomach,uterine. Administration is not limited to the treatment of an existingtumor or infectious disease but can also be used to prevent or lower therisk of developing such diseases in an individual, i.e., forprophylactic use. Potential candidates for prophylactic vaccinationinclude individuals with a high risk of developing cancer, i.e., with apersonal or familial history of certain types of cancer.

c. Immunosuppressed Conditions

The CNT compositions may also be used for treatment of diseaseconditions characterized by immunosuppression, including, but notlimited to, AIDS or AIDS-related complex, idiopathic immunosuppression,drug induced immunosuppression, other virally or environmentally-inducedconditions, and certain congenital immune deficiencies. The CNTcompositions may also be employed to increase immune function that hasbeen impaired by the use of radiotherapy of immunosuppressive drugs(e.g., certain chemotherapeutic agents), and therefore can beparticularly useful when used in conjunction with such drugs orradiotherapy.

d. Subjects Exposed to Allergens

The compositions and methods disclosed herein are useful to treat and/orpreventing allergic reactions, such as allergic reactions which lead toanaphylaxis. Allergic reactions may be characterized by the T_(H)2responses against an antigen leading to the presence of IgE antibodies.Stimulation of T_(H)1 immune responses and the production of IgGantibodies may alleviate allergic disease. Thus, the disclosed vaccinecompositions may lead to the production of antibodies that preventand/or attenuate allergic reactions in subjects exposed to allergens.These can be used to enhance blocking or tolerance inducing reactions.

e. Subjects with or at Risk of Developing Autoimmune Diseases orDisorders

The compositions and methods are useful for the treatment or preventionof autoimmune diseases and disorders. Exemplary autoimmune diseasesinclude vasculitis, Wegener's granulomatosis, Addison's disease,alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet'sdisease, celiac disease, chronic fatigue syndrome, Crohn's disease,ulcerative colitis, type I diabetes, fibromyalgia, autoimmune gastritis,Goodpasture syndrome, Graves' disease, idiopathic thrombocytopenicpurpura (ITP), lupus, Meniere's multiple sclerosis, myasthenia gravis,pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoidarthritis, rheumatic fever, sarcoidosis, scleroderma, vitiligo,vasculitis, small vessel vasculitis, hepatitis, primary biliarycirrhosis, rheumatoid arthritis, Chrohn's disease, ulcerative colitis,sarcoidosis, scleroderma, graft versus host disease (acute and chronic),aplastic anemia, and cyclic neutropenia.

f. Subjects Undergoing or at Risk of Graft Rejection orGraft-Versus-Host Disease

The compositions and methods are useful for the treatment or preventionof graft rejection or graft versus host disease. The methods andcompositions can be used in the prevention or treatment of any type ofallograft rejection or graft versus host disease for any type of graft,including a xenograft. The allograft can be an organ transplant, suchas, but not limited to, a heart, kidney, liver, lung or pancreas.Alternatively, the allograft can be a tissue transplant, such as, butnot limited to, heart valve, endothelial, cornea, eye lens or bonemarrow tissue transplant. In yet other embodiments, the allograft can bea skin graft.

B. Adoptive Immunotherapy

The disclosed CNT aAPCs and CNPs are particularly useful to activate Tcells ex vivo for adoptive immunotherapy. In adoptive immunotherapy, asource of T cells is obtained from a subject to be treated for use inadoptive immunotherapy in an organism in which an immune response can beelicited, e.g., mammals. Examples of subjects include humans, dogs,cats, mice, rats, and transgenic species thereof, although humans arepreferred. T cells can be obtained from a number of sources, includingperipheral blood leukocytes, bone marrow, lymph node tissue, spleentissue, and tumors. In a preferred embodiment, peripheral bloodleukocytes are obtained from an individual by leukopheresis. To isolateT cells from peripheral blood leukocytes, it may be necessary to lysethe red blood cells and separate peripheral blood leukocytes frommonocytes by, for example, centrifugation through, e.g., a PERCOLL™gradient.

A specific subpopulation of T cells, such as CD4⁺ or CD8⁺ T cells, canbe further isolated by positive or negative selection techniques. Forexample, negative selection of a T cell population can be accomplishedwith a combination of antibodies directed to surface markers unique tothe cells negatively selected. One suitable technique includes cellsorting via negative magnetic immunoadherence, which utilizes a cocktailof monoclonal antibodies directed to cell surface markers present on thecells negatively selected. For example, to isolate CD4⁺ cells, amonoclonal antibody cocktail typically includes antibodies to CD14,CD20, CD11b, CD16, HLA-DR, and CD8. The process of negative selectionresults in an essentially homogenous population of the desired T cellpopulation.

CNT aAPCs and CNPs may be customized according to the subject and thecondition or disease to be treated. In one embodiment, the CNT aAPCs andCNPs contain at least one polyclonal T cell receptor activator, such asan anti-T cell receptor antibody. Polyclonal T cell activation can beuseful because it can expand a T cell population more quickly thanantigen-specific methods. The expanded polyclonal T cells can then besorted to select for T cells with a specificity for the epitopes ofinterest. In another embodiment, the CNT aAPCs contain MHC class I orMHC class II molecules bound to antigens of interest forantigen-specific T cell activation. The MHC polypeptides used in the CNTaAPCs and CNPs are preferably selected to match the MHC allelesexpressed by the subject to be treated. The antigen is selected based onthe condition or disease to be treated or prevented. The antigen may bederived from the subject to be treated.

The selected T cells are then contacted in appropriate medium with theCNT aAPCs or CNPs. CNT aAPCs or CNPs are used in amounts effective tocause activation and proliferation of T cells. The T cells are contactedwith the CNT aAPCs or CNPs for periods of time necessary for expansionof the T cells. It may be advantageous to maintain long-term culture ofa population of T cells following the initial activation andstimulation, by separating the T cells from the stimulus after a periodof about 12 to about 14 days. In certain embodiments, it may bedesirable to separate the T cells from the stimulus after a period ofabout 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 days. In certain embodiments, itmay be desirable to separate the T cells from the stimulus after aperiod of less than one day, such as after about an hour, or 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23hours. The rate of T cell proliferation is monitored periodically (e.g.,daily) by, for example, examining the size or measuring the volume ofthe T cells, such as with a Coulter Counter. In this regard, a resting Tcell has a mean diameter of about 6.8 microns, and upon initialactivation and stimulation, in the presence of the stimulating ligand,the T cell mean diameter will increase to over 12 microns by day 4 andbegin to decrease by about day 6. The T cells may be stimulated throughmultiple rounds of activation by the CNT aAPCs or CNPs. For example,when the mean T cell diameter decreases to approximately 8 microns, theT cells may be reactivated and re-stimulated to induce furtherproliferation of the T cells. Alternatively, the rate of T cellproliferation and time for T cell re-stimulation can be monitored byassaying for the presence of cell surface molecules, such as, CD154,CD54, CD25, CD137, CD134, which are induced on activated T cells.

Following activation and expansion of the T cells, they are administeredto the subject in amounts effective to induce an immune response. Insome embodiments the T cells are isolated from CNPs prior toadministration to the subject, preferably using magnetic separation. TheT cells may be administered separately from, or in combination with, theCNT aAPCs or CNPs. The immune response induced in the animal byadministering the compositions may include cellular immune responsesmediated by CD8⁺ T cells, capable of killing tumor and infected cells,and CD4⁺ T cell responses. Humoral immune responses, mediated primarilyby B cells that produce antibodies following activation by CD4⁺ T cells,may also be induced. In a preferred embodiment, the immune response ismediated by cytolytic CD8⁺ T cells. A variety of techniques which arewell known in the art may be used for analyzing the type of immuneresponses induced by the compositions and methods disclosed herein(Coligan et al., Current Protocols in Immunology, John Wiley & Sons Inc.(1994)).

1. Adoptive Immunotherapy of Autoimmune Diseases and Disorders, AllergicReactions, Graft Rejection and Graft-Versus-Host-Disease

Adoptive immunotherapy may also be used to treat or prevent conditionsassociated with undesirable activation, over-activation or inappropriateor aberrant activation of an immune response, as occurs in conditionsincluding autoimmune disorders and diseases, allergic reactions, graftrejection and graft-versus-host disease. In one embodiment, undesirableor aberrant antigen-specific immune responses are treated or preventedby adoptive immunotherapy using “regulatory” T cells (Tregs) activatedby the compositions and methods disclosed herein.

Immunological self-tolerance is critical for the prevention ofautoimmunity and maintenance of immune homeostasis. The ability of theimmune system to discriminate between self and non-self is controlled bymechanisms of central and peripheral tolerance. Central toleranceinvolves deletion of self-reactive T lymphocytes in the thymus at anearly stage of development (Rocha, et al., Science, 251:1225-1228(1991); Kisielow, et al., Nature, 333:742-746 (1988)). Severalmechanisms of peripheral tolerance have been described, including T cellanergy and ignorance (Schwartz, Science, 248:1349-1356 (1990); Miller,et al., Immunol. Rev., 133:131-150 (1993)). Studies have provided firmevidence for the existence of a unique CD4⁺CD25⁺ population ofprofessional regulatory/suppressor T cells that actively and dominantlyprevent both the activation as well as the effector function ofautoreactive T cells that have escaped other mechanisms of tolerance(Sakaguchi, et al., J. Immunol., 155:1151-1164 (1995); Takahashi, etal., Int. Immunol., 10:1969-1980 (1998); Itoh, et al., J. Immunol.,162:5317-5326 (1999)). The elimination or inactivation of these cellsresulted in severe autoimmune disease, and was also found to enhanceimmune responses to alloantigens and even tumors (Sakaguchi, et al., J.Immunol., 155:1151-1164 (1995); Itoh, et al., J. Immunol., 162:5317-5326(1999); Shimizu, et al., J. Immunol., 163:5211-5218 (1999)).Autoantigen-specific regulatory T (Treg) cells actively regulateautoimmunity and induce long term tolerance and have application as astrategy for inducing long-lived tolerance.

T cells are obtained from the subject to be treated as described above,and a Treg enriched cell population is obtained by negative and orpositive selection. An autoantigen-specific regulatory T (Treg) cellenriched composition is one in which the percentage ofautoantigen-specific Treg cells is higher than the percentage ofautoantigen-specific Treg cells in the originally obtained population ofcells. In particular embodiments, at least 75%, 85%, 90%, 95%, or 98% ofsaid cells of the composition are autoantigen-specific regulatory Tcells. To maximize efficacy, the subpopulation is enriched to at least90%, preferably at least 95%, and more preferably at least 98% Tregcells, preferably CD4⁺CD25⁺CD62L⁺ Treg cells. Positive selection may becombined with negative selection against cells comprising surface makersspecific to non-Treg cell types, such as depletion of CD8, CD11b, CD16,CD19, CD36 and CD56-bearing cells.

The Treg cells are activated in a polyclonal or antigen-specific mannerex vivo using the compositions, as described above, expanded, andadministered to the subject to be treated. In another embodiment, apopulation of T cells not enriched for Treg cells is activated andexpanded, and the Treg cells are selected from the expanded T cellpopulation using appropriate positive and/or negative selection.

Adoptive immunotherapy using Treg cells can be used for prophylactic andtherapeutic applications. In prophylactic applications, Treg cells areadministered in amounts effective to eliminate or reduce the risk ordelay the outset of conditions associated with undesirable activation,over-activation or inappropriate or aberrant activation of an immuneresponse, including physiological, biochemical, histologic and/orbehavioral symptoms of the disorder, its complications and intermediatepathological phenotypes presenting during development of the disease ordisorder. In therapeutic applications, the compositions and methodsdisclosed herein are administered to a patient suspected of, or alreadysuffering from such a condition associated with undesirable activation,over-activation or inappropriate or aberrant activation of an immuneresponse to treat, at least partially, the symptoms of the disease(physiological, biochemical, histologic and/or behavioral), includingits complications and intermediate pathological phenotypes indevelopment of the disease or disorder. An amount adequate to accomplishtherapeutic or prophylactic treatment is defined as a therapeutically-or prophylactically-effective amount.

With respect to allograft rejection or graft versus host disease, in apreferred embodiment, adoptive immunotherapy with Treg cells isinitiated prior to transplantation of the allograft. In certainembodiments, the Treg cells can be administered to the subject for aday, three days, a week, two weeks or a month prior to atransplantation. In other embodiments, the Treg cells are administeredfor a week, two weeks, three weeks, one month, two months, three monthsor six months following a transplantation. In a preferred embodiment,Treg cells are administered both before and after a transplantation iscarried out.

The outcome of the therapeutic and prophylactic methods disclosed hereinis to at least produce in a patient a healthful benefit, which includes,but is not limited to, prolonging the lifespan of a patient, delayingthe onset of one or more symptoms of the disorder, and/or alleviating asymptom of the disorder after onset of a symptom of the disorder. Forexample, in the context of allograft rejection, the therapeutic andprophylactic methods can result in prolonging the lifespan of anallograft recipient, prolonging the duration of allograft toleranceprior to rejection, and/or alleviating a symptom associated withallograft rejection.

In another embodiment, undesirable or aberrant antigen-specific immuneresponses are treated or prevented by adoptive immunotherapy by usingthe compositions to activate and expand T cells specific for IgE orCD40L.

Immune responses to foreign, sometimes innocuous, substances such aspollen, dust mites, food antigens and bee sting can result in allergicdiseases such as hay fever, asthma and systemic anaphylaxis. Immuneresponses to self-antigens such as pancreatic islet antigens andcartilage antigens can lead to diabetes and arthritis, respectively. Thehallmark of the allergic diseases is activation of CD4⁺ T cells and highproduction of IgE by B cells, whereas the salient feature of autoimmunediseases are activation of CD4⁺ T cells and over production ofinflammation cytokines. Activated CD4⁺ T cells transiently express theself antigen CD40L.

Cytotoxic T lymphocytes (CTLs) specific for antigenic peptides derivedfrom IgE molecule can be generated ex vivo using the artificial antigenpresenting cells and methods disclosed herein presenting antigenic IgEpeptides. These IgE specific CTLs can be administered to a subject tolyse the target cells loaded with IgE peptides and inhibit antigenspecific IgE responses in vivo. These IgE specific CTLs can also be usedto prevent or treat the development of lung inflammation and airwayhypersensitivity.

Similarly, cytotoxic T lymphocytes (CTLs) specific for antigenicpeptides derived from CD40L can be generated ex vivo using theartificial antigen presenting cells and methods disclosed hereinpresenting antigenic CD40L peptides. These CD40L specific CTLs can beadministered to a subject to lyse target activated CD4⁺ cells in vivo.These CD40L specific CTLs can be used to inhibit CD4-dependent antibodyresponses of all isotypes in vivo.

C. Active Immunotherapy

The CNT aAPCs, CNPs, and vaccine compositions may also be used foractive immunotherapy. For active immunotherapy, the CNT aAPCs, CNPs, orCNT vaccine compositions are administered directly to the subject to betreated. In general, methods of administering vaccines are well known inthe art. Any acceptable method known to one of ordinary skill in the artmay be used to administer a formulation to the subject. Theadministration may be localized (i.e., to a particular region,physiological system, tissue, organ, or cell type) or systemic. CNTaAPCs, CNPs, or CNT vaccine compositions may be administered by a numberof routes including, but not limited to, injection: intravenous,intraperitoneal, intramuscular, or subcutaneous, to a mucosal surface(oral, sublingual or buccal, nasal, rectal, vaginal, pulmonary), ortransdermal. In some embodiments, the injections can be given atmultiple locations. The CNT aAPCs, CNPs, or CNT vaccine compositions canalso be administered directly to an appropriate lymphoid tissue, such asthe spleen, lymph nodes or mucosal-associated lymphoid tissue.

The CNT vaccine compositions are particularly suitable for enteraladministration. The ability to target vaccine compositions to epithelialcells in the digestive tract greatly facilitates the ability of avaccine to induce mucosal and systemic immunity when administeredorally. Molecules, as described above, which protect the vaccinecomposition and its constituents from hydrolysis and degradation in lowpH environments also enhance the efficacy of vaccines administeredorally.

Administration of the formulations may be accomplished by any acceptablemethod which allows an effective amount of the CNT aAPCs, CNPs, or CNTvaccine compositions to reach their target. The particular mode selectedwill depend upon factors such as the particular formulation, theseverity of the state of the subject being treated, and the dosagerequired to induce an effective immune response. As generally usedherein, an “effective amount” is that amount which is able to induce animmune response in the treated subject. The actual effective amounts ofcompositions can vary according to factors including the specificantigen or combination thereof being utilized, the density and/or natureof the associated co-stimulatory molecules, the release characteristicsof, the particular composition formulated, the mode of administration,and the age, weight, condition of the subject being treated, as well asthe route of administration and the disease or disorder.

EXAMPLES Example 1. Effect of Chemical Treatments on Surface Area ofSWNTs and Protein Adsorption

Materials and Methods:

Materials

Biotin Anti-CD3 was purchased from BD Biosciences—Pharminogen (San Jose,Calif.). Treated and raw SWNT were obtained from the Department ofChemical Engineering at Yale University.

Raw SWNT Chemical Treatment

SWNT bundles were stirred in a 3M HNO₃ at 70° C. for 1 hour. The samplewas then filtered through a 5 μm pore size PTFE membrane and dried at45° C. overnight in an oven. Reduced samples entailed the addition ofLiBH₄ solution in THF (200 mg SWNT+125 ml THF+400 mg LiBH₄), thensonication for 1.5 hour. This chemical procedure gives the (3MHNO₃/LiBH₄) SWNT group. If the procedure was stopped after the oxidationstep (without LiBH₄ treatment), the SWNT group is simply called (3MHNO₃).

Determination of Dry SWNT Surface Area

Dry SWNT (or A.C.) surface area was determined by physisorption ofnitrogen using the Brunauer-Emmett-Teller method (B.E.T). SWNT (or A.C.)physisorption and estimation of surface area were performed using anAutosorb-1 from Quantachrome.

Transmission Electron Microscopy (TEM)

TEM images were obtained using a Philips Tecnai F12 TEM instrument. Onemg of pre-weighed SWNT (or A. C) was mixed in 10 ml of ethanol (ACS/USPgrade), and dispersed by ultra-sonication. A droplet of the SWNT/ethanol(or A.C./ethanol) suspension was then applied on a holey carbon coatedcopper TEM grid before air-drying.

Scanning Electron Microscopy (SEM)

SEM images were obtained on a XL-30 ESEM-FEG microscope from FEICompany. One hundred micrograms per milliliter of SWNT (or A.C.)solution dissolved in PBS was washed and resuspended in dionized water.The washing step was repeated three times using a micro-centrifuge at12,000 rpm for 8 minutes. Ten microliters of the washed solution wasapplied to 0.25 cm² of carbon tape mounted on an aluminum stub. The stubis placed at −80° C. for 2 hours and then lyophilized overnight using aLabonco Free Zonel Lyophilizer (vacuum at 0.057 mBar, −44° C.collector).

SWNT Sterilization for Cellular Studies

A known amount of SWNT was dissolved in dionized water then heated in avacuum oven at 180° C. The SWNT sample was next sonicated in a knownvolume of sterile phosphate buffer solution (PBS) for 10 minutes thenexposed to ultraviolet light for 20 minutes. The same procedure wasapplied to activated carbon and C60 samples. PS samples were sterilizedusing a 0.45 um MILLEX®HA sterile filter unit (MF-Millipore MCEmembrane), dissolved in sterile PBS, then exposed to ultraviolet lightfor 20 minutes.

Adsorption of BSA onto SWNT

Bovine Serum Albumin (40 kDa) was selected as a model protein toquantify physical adsorption onto SWNT of known dry SWNT surface area. Asample obtained from a sterile stock of SWNT was dissolved in PBS to aconcentration of 300 μg/ml then sonicated for 10 minutes to obtainuniform dispersion. BSA at 600 μg/ml was serially diluted at 200 μl in1×PBS. The SWNT sample was then dispensed at an equal volume of 200 μlinto the prepared BSA samples. The mixture was allowed to mix in arotary shaker at 4° C. overnight. SWNT mixtures were then centrifuged ina micro-centrifuge at 12,000 rpm for 20 minutes. The supernatant wasremoved and analyzed for protein content using the BCA and the micro-BCAassays. The amount of BSA loaded onto SWNT was deduced from a simplemass balance based on the difference in protein concentration before andafter SWNT addition.

Results:

The tunability of protein adsorption on SWNT through chemical treatment,which may affect surface area for available protein interaction, wasfirst examined. Protein adsorption isotherms were compared fromuntreated SWNTs, SWNTs treated with a 3M nitric acid (3M HNO₃), andSWNTs treated with 3M nitric acid then reduced in lithium borohydride(3M HNO₃/LiBH₄). These treatments were chosen because refluxing SWNTs innitric acid introduces carboxylic acid groups at the open ends leadingto sites of defects and hence enhancing capacity for protein adsorption(Hu, et al., Jour. Phys. Chem. B, 107:13838-42 (2003)). A second stepinvolving the reduction of the carboxylic groups in lithium borohydridewill preferentially reduce the oxygenated groups created by previousacid treatment favoring the dispersion of SWNTs in solution (U.S.Published Application 2004/0232073) and further increasing surface areaavailable for protein adsorption.

Untreated and chemically treated SWNTs were examined at highmagnification under transmission electron microscopy (TEM). Aggregatednanotube structures consistent with the bundling morphology wereobserved. The three groups of SWNTs showed similar structural integrityalthough some differences were noted. First, the surface of nitric acidtreated SWNT appeared to be slightly damaged when compared to untreatedSWNT, and can be correlated with the observed increase in the bundlesurface area as measured by physiosorption (Hemraj-Benny, et al., Jour.Coll. Interf. Sci., 317(2):375-82 (2008); Cinke, et al., Chem. Phys.Lett., 365:69-74 (2002)). Second, the nitric acid treatment appeared toreduce the amount of impurities, such as remaining catalysts presentalong with the nanotubes (Hu, et al., Jour. Phys. Chem. B, 107:13838-42(2003); Hemraj-Benny, et al., Jour. Coll. Interf. Sci., 317(2):375-82(2008); Cinke, et al., Chem. Phys. Lett., 365:69-74 (2002); Park, etal., Journ. Mater. Chem., 16:141-54 (2006)) as seen by the reduction ofmetallic particles in the TEM images. This purification step may play animportant role in rendering the SWNT cytocompatible. The describedchemical treatments do not induce gross morphological changes to theSWNT bundles. All three groups appeared as porous curved surfaces underscanning electron microscopy, with crevices on the length scale ofcells, which may facilitate cellular interactions.

To ascertain the effect of these treatments on the surface area of thebundles, surface area was estimated using nitrogen physisorption(Hemraj-Benny, et al., Jour. Coll. Interf. Sci., 317(2):375-82 (2008);Cinke, et al., Chem. Phys. Lett., 365:69-74 (2002); Thommes, et al.,Langmuir, 22:756-64 (2006)). As expected, the surface areas derived fromFIG. 5 using Brunauer-Emmett-Teller (B.E.T) analysis, and summarized inTable 1, changed with the associated chemical treatment.

TABLE 1 Surface area of untreated and treated SWNTs SWNT Group Area(m²/g) Untreated 845 3M HNO₃ 1190 3M HNO₃/LiBH₄ 1560Untreated SWNTs have the least surface area at 845 m²/g. SWNTs treatedwith 3M HNO₃ produced a surface area of 1190 m²/g. Finally, SWNTstreated with 3M HNO₃/LiBH₄ produced the largest increase in surface areaat 1560 m²/g. The reduction step with LiBH₄ that follows the 3M HNO₃treatment may also play a role in enhancing SWNT surface area furtherthrough de-bundling (Liang, et al., Nano Lett., 4:1257-60 (2004)),although such a mechanism is not completely understood.

To assess the effects of chemical treatment of SWNT bundles on proteinadsorption, treated and untreated SWNTs were incubated with a modelprotein, bovine serum albumin (BSA) and measured the respectiveadsorption isotherms. Results are shown in FIG. 6. The adsorption curvesshow a correlation between the type of chemical treatment and maximalprotein adsorption. SWNTs treated with 3M HNO₃/LiBH₄ produced thehighest measured surface area and subsequent higher protein adsorption.Thus, this treatment was selected for further studies on T cellstimulation.

Example 2. Cytocompatibility of SWNTs

Materials and methods were as described above with respect to Example 1,except as noted below.

Materials and Methods:

Cytotoxicity Study

A metabolic assay, Cell Titer Blue (CTB) assay, was used to assess cellviability exposed to SWNT. Each group was cultured in triplicates. Ameasured amount of sterile SWNT at 2.5 mg/ml was serially diluted in asterile 96 well U-bottom cell culture plate with RPMI 1640 containing10% fetal bovine serum (FBS) and 2% penicillin streptomycin (PS). Sodiumazide was included as a negative control at an initial volume of 2.5%and was diluted analogously. Hybridoma T cells (B3Z) at a concentrationof 4×10⁵ cells/ml were then added uniformly to the assay. The plate wasincubated for 24 hours at 37° C. and 5% CO₂. Forty microliters of CTBproliferation reagent was added to each well, including the control (noSWNT) then incubation was allowed for 4 more hours at 37° C. and 5% CO₂.Cell proliferation was monitored with the use of cellular standard ofknown cell population. Fluorescence was recorded at an excitation of 560nm and emission of 590 nm using a Spectra Max M5 spectrometer fromMolecular Devices (Sunnyvale, Calif.).

Results:

The cytocompatibility of SWNTs modified with 3M HNO₃/LiBH₄ on T cellswas examined next. SWNTs from this treatment group were titrated andincubated with T cells for 24 hours before comparison to a cytotoxiccontrol, sodium azide. It was observed that treated SWNT did not presentany significant toxic effects on T cells below a concentration of 150μg/ml (FIG. 7). This minimal toxic effect observed with the treatednanotubes was not unexpected since the overall length scale of SWNTbundles for cellular interaction is significantly larger than otherreports which support SWNT toxicity Magrez, et al., Nano Lett., 6:1121-5(2006); Porter, et al., Nature, 2:713-7 (2007)). In these reports,single tubes (significantly smaller then bundles) may be internalizedcausing the observed toxic effects. Thus, internalization of SWNT by Tcells is improbable as length of bundles is on the order of hundreds ofnanometers to microns. A second possible reason for the observed minimaltoxicity is the fact that chemical treatment of SWNTs dissolves themajority of remaining impurities and metal catalysts from previousreactions. This step could provide an improved environment for theproliferation of cells Porter, et al., Nature, 2:713-7 (2007); Shvedova,et al., Jour. Tox. Env. Hlth. Pt. A, 66(20):1909-26 (2003)). Also, thetime scale needed for appropriate activation of T cells is significantlyshorter than most time scales involved in reported toxicity results.Finally, the relatively high solubility of 3M HNO₃/LiBH₄ SWNTs in watercould play a role in enhancing cytocompatibility (Dumortier, et al.,Nano Lett., 6:1522-8 (2006)).

Example 3. Stimulation of T Cells Using SWNTs

Materials and methods were as described above with respect to Examples 1and 2, except as noted below.

Materials and Methods:

T-Cell Stimulation Using Anti CD3 Loaded SWNT

The stimulation of T-cells (B3z) was quantified using a mouse IL-2ELISA. Anti-CD3 and SWNT bundles were mixed overnight at 4° C. on arotary mixer to allow for physical adsorption of anti-CD3, followed by awashing step to remove unbound anti-CD3. The washed sample was thenre-suspended in sterile PBS at the same initial volume. SWNT antibodysolution was serially diluted in a microplate followed by the additionof 100 μl of T-Cells (B3z) at 4×10⁵cells/ml. The cell culture plate wasincubated for 24 hrs. at 37° C. and 5% CO₂. An identical protocol wasused for all SWNT groups. For control, soluble and plated anti-CD3, thestimulus was used in similar amounts to anti CD3 loaded SWNT before thewash step. After incubation an ELISA was then performed on IL-2extracted from the supernatant of each well to quantify the stimulationof T cells.

Fluorescence Imaging of T-Cell Stimulation Using Anti-CD3 Loaded SWNT

Cells were labeled with carboxyfluorescein diacetate-succinimide ester(CFDA-SE) according to established protocols (Invitrogen-Molecularprobes). 1 μl of CFDA-SE was added to cells for 15 minutes at 37° C. and5% CO₂. Cells were then centrifuged and washed 3× in cold 1×PBS. Stainedcells were washed once with PBS then added in along with anti CD3 loadedSWNT for stimulation (as described). Imaging was performed using anOlympus IX71 microscope and QICAM 32-0030C-152 camera from QImaging.

Results:

Stimulation of T cells using anti-CD3 adsorbed onto 3M HNO₃/LiBH₄ SWNTwas investigated and compared this stimulation to anti-CD3 immobilizedon tissue culture plate or free in solution. SWNT bundles incorporatingthe anti-CD3 stimulus had a dramatic effect on T cell activation asmeasured by the release of IL-2 (FIG. 8). Activation with antibodyimmobilized on SWNT was at least four-fold and six-fold greater incomparison to plate-bound antibodies and soluble antibodiesrespectively. This was consistent with a model concentration-responsefit which suggested that the concentration of antibody at whichhalf-maximal T cell stimulation takes place was significantly lower forantibody-SWNT combinations versus plate bound or soluble antibody (Table2).

TABLE 2 LOG [EC50] for T cell stimulation by various compositions LOG[EC50] R-squared Figure 8 3M HNO₃/LiBH₄ (+) 0.036 ± 0.002 0.977 PB(anti-CD3) 0.295 ± 0.020 0.952 Sol (anti-CD3) 0.213 ± 0.201 0.937 Figure9 3M HNO₃/LiBH₄ 0.090 ± 0.002 0.981 3M HNO₃ 0.278 ± 0.019 0.929Untreated 0.973 ± 0.081 0.830 Sol (anti-CD3) N/A N/A Figure 10 100 μg/ml(+) 0.039 ± 0.002 0.975 50 μg/ml (+) 0.080 ± 0.004 0.971 25 μg/ml (+)0.128 ± 0.005 0.979 12.5 μg/ml (+) 0.250 ± 0.015 0.951 Figure 11 SWNT0.088 ± 0.004 0.980 (3M HNO₃/LiBH₄) A.C. (untreated) 2.286 ± 0.165 0.735A.C. (3M HNO₃) 1.945 ± 0.104 0.876 A.C. N/A N/A (3M HNO₃/LiBH₄) Figure12 SWNT 5.904E−4 ± 0.001   0.980 (3M HNO₃/LiBH₄) SWNT (3M HNO₃) 2.242E−3± 0.001   0.931 SWNT (untreated) 7.285E−3 ± 0.001   0.913 A.C.(untreated) 5.830E−3 ± 0.001   0.816 A.C. (3M HNO₃) 0.0178 ± 0.001 0.939 PS-OH  2.091 ± 15.980 0.889 PS-COOH 0.291 ± 0.061 0.906 C60 N/AN/A C60-OH N/A N/AIt was hypothesized that the enhanced stimulation is due to cellularaggregation on the SWNT-stimulus system. Preferential aggregation of Tcells onto 3M HNO₃/LiBH₄ SWNT during stimulation was confirmedqualitatively by comparing the cellular proliferation of fluorescentlylabeled T cells around anti-CD3 immobilized on SWNT versus blank SWNT.In these images, selective aggregation of T cells was observed aroundanti-CD3 adsorbed onto 3M HNO₃/LiBH₄ SWNT scaffolds when compared to 3MHNO₃/LiBH₄ SWNT alone.

To determine the effect of surface treatment on antibody adsorption andlevels of T cell stimulation, anti-CD3 adsorbed onto treated anduntreated SWNT bundles was incubated with lymphocytes and T cellactivation was measured (FIG. 9). The response observed in 3M HNO₃/LiBH₄SWNT was more pronounced than other SWNT groups. Modeling parametersfrom Table 2 show that half maximal stimulation correlated with thisobservation. The Log [EC50] value for 3M HNO₃/LiBH₄ SWNT was thesmallest (0.090) when compared to other groups (0.278 for 3M HNO₃ and0.973 for untreated). Chemical treatments alter surface functionalgroups as well as increase the surface area of SWNT bundles (FIG. 5). Tcell activation by antibody adsorbed to functionalized 200 nmpolystyrene nanoparticles (PS) indicates however that T cell stimulationis not affected by altering surface chemistry from carboxylate groups(mimicking 3M HNO₃ treatment) or hydroxyl groups (LiBH₄ reduction afteroxidation). Thus, the vast surface area of 3M HNO₃/LiBH₄-treated SWNTbundles is primarily responsible for the observed increase in T cellstimulation. Additionally, these data demonstrate that chemicaltreatment can be used to tune the extent of protein adsorption and, inturn, to control the degree of T cell stimulation.

Levels of T cell stimulation can be modulated by varying theconcentration of SWNT stimulus bundles and keeping the amount ofanti-CD3 constant during pre-treatment (FIG. 10). T cells stimulatedwith SWNT bundles were responsive to the density of SWNT in aconcentration-dependent manner. Model fits from Table 2 show acorrelation between the SWNT concentration and the half-maximal T cellresponse. This indicates that overall contact area facilitating a highdensity of antigen-presentation is a determinant factor for the observedefficiency of SWNT-stimuli on T cell stimulation.

Example 4. Comparison of SWNTs and Other High Surface Area Materials forT Cell Activation

Materials and Methods:

Materials and methods were as described above with respect to Examples1-3, except as noted below.

Surface Area Calculation of PS Beads

Particle count for both PS-OH and PS—COOH is 5.68×10¹² particles/ml(Polysciences Inc.). The particle diameter is 200 nm in both groups. Theoverall area for presentation was estimated at 2.9×10⁻³ m² (or 29 m²/g)for both PS groups.

Results:

To further investigate the effect of the SWNT surface area on T cellactivation, the stimulation potential of other high surface areamaterials after antibody adsorption was studied. Stimulation byactivated carbon (A.C.) (1762 m²/g) was first compared because of thesimilarity of this material to SWNT bundles. The surface area of theactivated carbon was increased with SWNT bundles, but 3M HNO₃ treatmentresulted in a reduction (Nyogi, et al., Acc. Chem. Res., 35(12):1105-13(2002)) of the overall surface area (down to 573 m2/g) and 3M HNO₃/LiBH₄treatment yielded a sample with an indeterminate surface area (see Table3).

TABLE 3 Surface area of untreated and treated activated carbon (A.C.)A.C. Group Area (m²/g) Untreated 1762 3M HNO₃ 573 3M HNO₃/LiBH₄ N/A

As shown in FIG. 11, stimulation with all activated carbon samples iswell below that of 3M HNO₃/LiBH₄-treated SWNT bundles. However, the datain FIG. 11 do not account for surface area differences. Thus, antibodypresentation was normalized by surface area in 9. This figureadditionally shows stimulation by 200 nm polystyrene beads (29 m2/g),C60 and hydroxylated C60 (C60-OH; both estimated (Vogel, et al., Appl.Phys. A—Mater. Sci. Proc., 62:295-301 (1996)) at 4 m²/g). Despite thehigh surface area of all materials, SWNT bundles on an antibody per areabasis displayed the highest activation potential, as demonstrated by theLog [EC50] values (see Table 3), and in agreement with the proteinadsorption superiority of this material. These data support the uniquecapability of SWNT bundles to enhance T cell stimulation.

Example 5. Use of SWNTs as Vaccine Adjuvants

Adjuvants are critical components of vaccines. When mixed with anantigen, they enhance the antigen-specific immune response afterimmunization. Currently, there are a limited number of adjuvantsapproved for clinical use (aluminum based salts and a squalene-oil-wateremulsion MF59). Adjuvants can activate antigen-presenting cells (APCs)to stimulate T cells more efficiently, natural killer cells (NK cells),or other cells of the innate system to produce cytokines or promotesurvival of antigen-specific T cells.

In this study, single walled carbon nanotubes (SWNT) were assessed forpossible adjuvant properties using the model antigen ovalbumin (OVA).Recent studies have elucidated the potential of SWNT to activatecomplement via both classical and alternative pathways, as well as theircapacity to provoke inflammation and granuloma formation(Salvador-Morales, et al., Molecular Immunology, 43:193-201 (2006)).

C57BL6 mice were treated subcutaneously with either OVA alone, with OVAadsorbed to alum, with OVA adsorbed onto SWNT bundles, or withphosphate-buffered saline as a vehicle control. Details of theexperimental conditions are provided in Table 4.

TABLE 4 Experimental conditions for vaccination Treatment Group DoseSWNT + OVA 100 μg in 0.3 ml of PBS adsorbed to 25 μg/ml SWNT per mouseAlum + OVA 1 μl of Alum per 1 μg of OVA, 0.3 ml Tris buffer OVA alone100 μg OVA in 0.3 ml of PBS per mouse PBS 0.3 ml PBS per mouseThree mice were used for each condition. Serum was collected from allanimals prior to dosing. Mice were injected subcutaneously and wereeuthanized 10 days later for collection of serum and spleens.

OVA specific antibody T cell responses were analyzed by isolation ofspleenocytes from animals post euthanization. Spleenocytes werechallenged with OVA to assess cellular immune induction. Induction wasdetermined by measuring the production of 11-2 by the spleenocytes.Cellular immunity was positive for the SWNT OVA group across a range oftitrated antigen (FIG. 13). Responses of spleenocytes from mice from allother treatment groups was lower, including from the mice treated withAlum, which is a gold standard adjuvant.

Similarly, when mice were immunized intraperitoneally and orally withOVA adsorbed to SWNT bundles, T cells obtained from the immunized micedemonstrated strong induction of IL-2 in response to challenged with OVAat 1 mg/ml (FIG. 14).

Example 6. Design and Characterization of CNPs for Clustered AntigenPresentation, IL-2 Delivery, and Magnetic Enrichment of Activated TCells

Materials and Methods

Carbon Nanotubes

Bundled CNTs were synthesized from cobalt-incorporated MCM-41, purifiedusing a four-step treatment procedure, oxidized in 3M nitric acid (SigmaAldrich, St. Louis, Mo.), then reduced in lithium borohydride (SigmaAldrich) as published previously. See Chen et al. (2007), ACS Nano1:327-336 and Fadel et al. (2008), Nano Letters 8:2070-2076. CNTsuspension (50 μg/mL) in Phosphate Buffer Saline (PBS) (ThermoScientific, Waltham, Mass.) was sonicated for 10 min to obtain uniformdispersion, and then re-suspended in a 0.8 □M solution of neutravidin(Invitrogen, Carlsbad, Calif.) in PBS to obtain ^(N)CNTs. The mixturewas allowed to mix in a rotary shaker at 4° C. overnight.

Synthesis of Magnetite

Iron acetylacetonate (Fe(acac)₃) (0.399 g, 1.56 mmol), oleic acid (1.50mL, 4.64 mmol), oleylamine (1.03 mL, 3.09 mmol), 1,2-hexadecanediol(2.03 g, 7.76 mmol) and benzyl ether (10 mL) (all obtained from SigmaAldrich) were added to a single-neck round bottom flask equipped with amagnetic stir bar and a condenser, and deoxygenated for 1 hr. Thereaction was gradually heated at 3° C./min to 200° C., and held at thattemperature for 3 hr before cooling at room temperature. A final blacksolution was observed. The reaction mixture was precipitated in ethanol(Sigma Aldrich) and centrifuged twice. Ethanol was decanted and theproduct was dried via nitrogen purge leaving a black powder.

Preparation of IL-2/Magnetite Co-Loaded Nanoparticles with BiotinSurface Functionality (NPs)

50 μL of recombinant proleukin human IL-2 (Novartis, East Hanover, N.J.)at 1.2 mg/mL in PBS was added dropwise to a vortexing solution ofPoly(D,L-lactide-co-glycolide) (PLGA) 50:50 (100 mg) with an inherentviscosity of 0.59 dL/g (Lactel Polymers, Inc. Pelham, Ala., USA) andhydrophobic magnetite (18 mg) dissolved in 2 mL chloroform (ThermoScientific). The mixture was added dropwise to a 3.2 mL of a vortexingsolution of 5% poly-vinyl alcohol (PVA, Sigma-Aldrich) with MW average30-70 kD and DSPE-PEG-Biotin (4.14 mg/0.828 mL) (Avanti Polar Lipid,Alabaster, Ala.). The resulting mixture was then sonicated 3 times for10 s at 38% amplitude (TEKMAR VCW 400 W). The solution was addeddropwise to 100 mL of 0.2% PVA, and left stirring for 3 hr to evaporatethe solvent. Particles were collected by centrifugation at 12,000 rpmfor 15 min at 4° C., then washed 3 times with de-ionized water. NPs werelyophilized and stored at −20° C. until use. NPs with varying amounts ofmagnetite were also prepared in identical fashion.

Transmission Electron Microscopy (TEM)

TEM examination NPs and gold-tagged neutravidin adsorbed on bundled CNTswere evaluated on a Tecnai T12 HR-TEM (FEI Company, Hillsboro, Oreg.)operating at 120 kV. Neutravidin was previously conjugated with astoichiometric amount of biotinylated gold nanoparticles with a diameterof 10 nm (NANOCS, New York, N.Y.). Digital electron micrographs wereacquired with a high-resolution 4 k×4 k GATAN Ultrascan CCD camera(Pleasanton, Calif.). A drop of well-dispersed particle suspension wasplaced on an EMS carbon film 400 mesh copper grid (E.M.S., Hatfield,Pa.), then dried at ambient conditions prior to placement in the sampleholder of the microscope.

Scanning Electron Microscopy (SEM)

XL-30 ESEM FEG (Philips, Andover, Mass.) was used to visualize bundledCNTs and CNPs. The particles were fixed on an aluminum stub using2-sided carbon tape (E.M.S). The samples were imaged using a LaBelectron gun with an accelerating voltage of 5-10 kV. Size distributionand average particulate diameter of bundled CNTs were determined byanalyzing approximately 142 particulates per image using the freewareprogram NIH ImageJ.

Nanoparticle Size and Separation Analysis

NanoSight (Amesbury, U.K.) with an NS500 Microscope and NTA 2.0 Softwarewas used to visualize and determine the size distribution,concentration, and degree of nanoparticles. NPs were dispersed in PBS atdilute concentrations prior to analysis (125 μg/mL). NanoSight's“Nanoparticle Tracking Analysis” (NTA) detects and visualizespopulations of nanoparticles in liquids and measures the size of eachparticle from direct observations of diffusion as well as concentrationin number of particles per milligram of sample. NPs were first purifiedimmediately after synthesis by a 10 min separation using a 0.5 T magnet.Separation of CNPs involved two 10 min periods of separation. Thesupernatant was poured out and analyzed by NanoSight or flow cytometry.

Inductively Couple Plasma Mass Spectrometer (ICP-MS)

The iron content of NPs were determined by using an inductively coupleplasma mass spectrometer (ICP-MS) (ELEMENT XR, Thermo Scientific). 56Fewas measured using medium resolution (4000) and high resolution (10000)mass/mass difference. Under these conditions, 40Ar16O, which only needsa resolution of ˜2500 (mass/mass difference), was readily distinguishedfrom 56Fe. Concentrated nitric acid and hydrogen peroxide (SigmaAldrich) were added to completely digest the samples to release iron foranalysis.

Controlled Release of IL-2

17.5 mg of NPs were transferred to 1.7 mL Eppendorf tubes, suspended in0.350 mL PBS and incubated at 37° C. on a rototorque for a week.Periodic sampling of the amount of IL-2 released from the nanoparticleswas performed by centrifuging the nanoparticles, removing 0.350 mL ofthe supernatant for analysis and the re-suspending the nanoparticles in0.350 mL of fresh buffer. Enzyme-linked immunosorbent assay (ELISA)analysis was performed to measure IL-2 levels according to themanufacturer's guidelines (BD Biosciences, San Jose, Calif.).

FRET Sample Preparation and Analysis

Donor-only (5 μg/mL), acceptor-only (5 μg/mL), and donor-acceptorsamples (2.5 μg/mL each) were prepared by incubating ^(N)CNTs (25 μg/ml)with goat anti-mouse IgG-2a tagged with Alexa Fluor 555 (AF555)(Molecular Probes Inc., Eugene, Oreg.) as the donor, goat anti-mouseIgG-2a tagged with Alexa Fluor 647 (AF647) (Molecular Probes Inc.) asthe acceptor, or an equimolar mixture of both. In each case, FRETantibodies were bound to stoichiometric amounts of biotinylatedanti-goat IgG (R&D Systems, Minneapolis, Minn.) before binding ^(N)CNTs.Samples were tumbled at room temperature for 1 hr, away from light.Samples were then washed twice in PBS. A fourth group of CNT without aFRET pair was included as a control. FRET acceptor photobleaching(FRET-AP) was performed using a Leica SP5 microscope (Allendale, N.J.),and images were processed using Leica LAS AF (including FRET efficiencyanalysis) and MATLAB as previously described in Fadel et al. (2010),Langmuir 26:5645-5654.

Results

CNTs were synthesized as described above, treated with 3M HNO₃, thenreduced in LiBH₄ to yield ultra-pure, hydroxyl-modified CNTs, which wererich in surface defects and presented a high surface area as previouslymeasured. See Fadel et al. (2008), Nano Letters 8:2070-2076; Fadel etal. (2010), Langmuir 26:5645-5654; and Fadel et al. (2012) Small. In thescheme shown in FIG. 2, a protein linker, neutravidin, was adsorbed toyield ^(N)CNTs, then stoichiometric amounts of biotinylated T cellstimulatory signals added. This simple method has been previouslydemonstrated to yield the stable presentation of physiological T cellantigens such as MHC-I. See Fadel et al. (2012) Small. A third crucialsignal in the design is local paracrine delivery of cytokines to Tcells. By binding NPs co-encapsulating IL-2 and magnetite to ^(N)CNTs,multivalent presentation of physiological T cell stimulatory signals wasintegrated with paracrine delivery of IL-2, and enabled the magneticseparation of CNPs from T cells (FIG. 3). This is tested by firststimulating ovalbumin-specific CD8⁺ T cells directly isolated fromtransgenic mice (OT-1), then measuring the therapeutic efficacy of theseactivated T cells in vivo in mice inoculated with melanoma cellsexpressing the ovalbumin antigen (B16-OVA) following magnetic separationfrom CNPs (FIG. 4).

TABLE 5 Physiochemical properties of two main components of CNP.Functionalized Property CNT NP Diameter ~0.8 nm/nanotube ~150-200nm/particle Length ~0.5-5 μm/tube N/A ~20-40 μm/assembly Zeta Potential−2.6 mV −2.2 mV (in H₂O) Zeta Potential −26.4 mV −5.0 mV (in buffer)Surface Group 1745 m²/g 5 · 10⁻¹³ m²/particle Functional HydroxylPEG-Biotin Group Magnetite N/A 12% by weight Loading of PLGA Protein ~8nmol of ~50 μg IL-2/ Loading neutravidin/ 100 mg PLGA mg CNTConcentration 5 μg/mL 125 μg/mL Used in Culture Amount of 2.1 □M N/AAntigen Presented

Progression of the platform from bundled CNTs into CNPs was observed bySEM and TEM. SEM and TEM images showed the presence of structural gapsin CNTs that are amenable for the adsorption of proteins, and SEM imagesof the successful integration of ^(N)CNTs with NPs. Encapsulation ofmagnetite in NPs and the qualitative binding of neutravidin-gold tobundled CNTs by TEM was confirmed. The size of the CNT bundles wasdetermined from the SEM images using ImageJ for 142 particles. The sizedistribution of the CNT bundles is plotted in FIG. 15. CNTs weredetermined to bundle into particles of −13 μm in diameter and werefunctionalized to yield surface defects, thus resulting in a highersurface area. The physiochemical properties of the CNTs and NPs aresummarized in Table 5. The size of the magnetic loaded NPs wasdetermined by nanoparticle tracking analysis. The distribution of NPsizes is plotted in FIG. 16. The diameter of NPs was estimated aroundapproximately 264 nm from this distribution. Biotin-PEG functionality onthe NP surface was confirmed by ¹H NMR spectroscopy with the presence ofthe methylene protons from the PEG segment appearing at approximately3.5 ppm. As expected, NPs exhibited superparamagnetic properties at roomtemperature as determined by superconducting quantum interference device(SQUID) analysis. The adsorption isotherm of neutravidin on CNTs alsoshowed saturation at 8 nmol of protein/mg CNT. Additional studiesdetermined that optimal T cell stimulation was achieved using 5 μg/ml ofCNT, 125 μg/ml of NP, and a 2.1 μM concentration of biotinylated MHC-Iand μCD28. Comparing CD8⁺ T cell response using avidin orstreptavidin-bound CNTs (^(A)CNTs and ^(S)CNTs, respectively) to^(N)CNTs in similar experimental conditions indicated that, although theamount and distribution of protein is the same in all three platforms,the charge microenvironment provided by ^(N)CNTs was more optimal forthe stimulation of T cells.

TABLE 6 Effect of magnetite loading on magnetic separation, IL-2 releaseand T cell stimulation Percent Loading of Magnetite in NP 6 wt % 12 wt %30 wt % % Separation 72.5 ± 4.9  97.7 ± 1.0 95.8 ± 1.2 Efficiency Il-2Released 773.5 ± 47.4 480.5 ± 9.2 293.9 ± 1.6  (pg/10¹⁰ particles) Tcell response 1622.5 ± 834.4 5056.3 ± 595.7 3122.5 ± 413.7 (pg IFN-γ/mL)

To investigate the impact of magnetite loading on IL-2 release from NPsand subsequent T cell stimulation, loading of magnetite by weightpercent (wt %) was varied between 6 wt %, 12 wt % and 30 wt %. Theresults are summarized in Table 6. First, the results indicate thatthere is a strong correlation between the wt % of magnetite and thenumber of NPs encapsulating magnetite. For example, doubling magnetiteloading from 6 wt % to 12 wt % results in half as many particles, closeto 0.8×10¹⁰ particles. Second, varying the wt % of magnetiteco-encapsulated with a same amount of IL-2 in NPs significantly affecteda seven-day cumulative release of cytokine on a-per-particle basis.Third, CD8⁺ T cells incubated with CNPs at various wt % loading ofmagnetite showed an optimal IFN-gamma response at day 3 to particlesloaded at 12 wt % using equal particle concentration in all threegroups. Finally, overall separation efficiency of T cells from CNPs wasmeasured to be above 95% at a magnetite loading of 12 and 30 wt %. Thus,by designing CNPs containing NPs loaded with 12 wt % magnetite, cellseparation efficiency was measured at approximately 98% using flowcytometry. The NP scatter profile located at low forward scatter (FSC)indeed decreased from 8.59% of gated population to 0.17% after magneticseparation. Images of samples before and after separation indicatedsuccessful separation of CNPs from cells. To visualize protein clusterformation on the surface of CNT bundles, secondary antibody pairs,either with acceptor or donor fluorescence probes each bound to abiotinylated primary antibody, were allowed to bind to ^(N)CNTs.Fluorescence images of both donor and acceptor channels were observedafter addition to ^(N)CNTs. Acceptor photobleaching of the antigen-bound^(N)CNT platform triggered an increase in donor emission. Therepresentative FRET efficiency map derived from the change in donoremission clearly pointed to the presence of several micron-scale antigenclusters. The FRET efficiencies were measured to be 60-70%. Finally, thepresentation of biotin on NPs was achieved using fatty acids and lipidsas hydrophobic anchors to strongly associate with the PLGA matrix. Thisbiotinylation further affords the attachment to the ^(N)CNT platform andallows co-encapsulation and prolonged retention of fatty acid-modifiedmagnetite. We measured the cumulative release of IL-2 and iron from NPsloaded with 12 wt % magnetite. The release profile of IL-2 and iron fromthe 12 wt % magnetite NPs is plotted in FIG. 17. The controlled releaseprofile of IL-2, measured over a period of more than a week, is typicalof protein release during PLGA degradation. See for example Steenblockand Fahmy (2008), Mol. Ther. 16:765-772 and Steenblock et al. (2011), J.Biological Chem. 286:34883-34892. It is characterized by an initialburst release followed by continual release of protein over time.Leaching of iron from NPs was negligible over 150 hours, and wasmeasured below 0.01% of total iron loaded in the particles.

Example 7. In Vitro Stimulations of CD8+ T Cell Effectors by CNPs

Material and Methods

Antigen-Specific T Cell Stimulation Studies.

OT-1 mice (in which CD8+ T cells express a transgenic TCR specific forthe SIIN peptide of ovalbumin presented on H-2K^(b)) were bred,maintained, and screened in the Malone Engineering Center at YaleUniversity. Splenocytes were isolated from the spleen of OT-1 mice (aged6-8 weeks) after depletion of erythrocytes by hypotonic lysis. CD8⁺ Tcells were isolated using a CD8⁺ negative selection kit (StemCell Inc.,Vancouver, Canada). CD8⁺ T cells were resuspended in cell media,composed of RPMI 1640 supplemented with FBS (10%), L-glutamine (1%),HEPES buffer (1%), non-essential amino acids, 2-ME (0.1%), penicillin(2%), and stored at 4° C. before use. Equimolar amount (2.1 nM total) ofbiotinylated H-2K^(b) loaded with SIINFEKL peptides (MHC-I) andanti-CD28 (BD Biosciences) were added to the ^(N)CNT suspension, andallowed to mix for 1 hr at room temperature. As a final step, NPs (625μg/mL) were added to the mixture and allowed to bind for ½ hour. Themixture was then diluted in cell media (1:5) then added to an equalvolume of CD8⁺ T cells (5×10⁵ cells/mL) in a 24-well plate. For controlgroups, DYNABEADS® (Invitrogen) were added at a final concentration of1×10⁶ particles/mL, in similar cell culture conditions to CNPs. Thecells were then incubated at 37° C. After three days of culture, sampleswere collected by group (including supernatant for cytokine analysis),purified using magnetic separation to isolate activated T cells,counted, and sized using a Multisizer 3 Coulter counter at a 1:400dilution (Beckman Coulter Inc., Indianapolis, Ind.), then processed foranalysis. For long-term expansion studies, T cells collected at day 3were re-stimulated using a same quantity of soluble MHC-I and αCD28,along with exogenous IL-2 or NPs depending on the groups. Cells wereactivated using CNPs for the first 3 days to avoid effects related to Tcell exhaustion and activation-induced cell death. At day 5, 7, and 14,T cells will be collected, purified using magnetic separation, countedand sized, and re-stimulated using similar conditions to day 3.

Immunofluorescence

After separation, CD8⁺ T cells were washed twice in a staining buffersolution (1% BSA in PBS), and adjusted at a concentration of 5×10⁶cells/mL. For cell surface staining, T cells were resuspended instaining solutions containing antibody mixtures at a ratio of 1:400 instaining buffer for 30 min at 4° C. A six-color combination was selectedusing the following antibodies: anti-CD44 FITC, anti-CD8 PacBlue,anti-CD25 AF700, anti-CD62L APC, anti-CD27 PE, anti-CD69 PE-Cy7(eBiosciences). After incubation, cells were washed twice in stainingbuffer then resuspended in 4% paraformaldehyde (PFA) (USB Corp.,Cleveland, Ohio) before measurement. For intracellular staining, CD8⁺ Tcells were incubated in the presence of GolgiPlug (BD Biosciences) for 4hr, washed twice in Perm/Wash™ solution (BD Biosciences), andresuspended in 250 μl of Cytofix/Cytoperm™ (BD Biosciences) solution for20 min at 4° C. T cells were washed twice in Perm/Wash™ solution thenresuspended in a staining solution containing anti-granzyme-B PE-Cy7(eBiosciences) at a ratio of 1:400 in staining buffer for 30 min at 4°C. After incubation, cells were washed twice in PERM/WASH™ thenresuspended in 4% PFA before measurement. Flow cytometry measurementswere performed using a Becton Dickinson LSR-II (San Jose, Calif.) withappropriate compensation and staining controls. Analysis of fluorescencewas performed using FlowJo software (Tree Star, Ashland, Oreg.) andgating on CD8⁺ subsets in side scatter (SSC) vs. forward scatter (FSC)plots. Granzyme-B mean fluorescence was normalized to the meanfluorescence measured in CNP-activated T cells.

Imaging of CD8⁺ T Cells

Expansion of CD8⁺ T cells was imaged using an inverted phase-contrastEclispse TS100 microscope (Nikon Inc., Melville, N.Y.) in a 24-wellplate at 24, 48, and 72 hr of culture. 20× and 100× images were capturedusing a high-resolution digital camera DXM1200F (Nikon, Melville, N.Y.).Interaction of T cells with CNPs was imaged using a Leica SP5 confocalmicroscope and analyzed using Leica LAS AF. T cells were cultured inpresence of CNPs (or appropriate controls) in 4-well Lab-Tek™ chamberslide system (Thermo Scientific) as described above. After 24 hr ofculture, the slide system was centrifuged at 1500 rpm, washed twice instaining buffer. Cell surface staining was performed using CD25-FITC(eBiosciences) at 1:400 in staining buffer for 1 hr at room temperatureaway from light. The slide system was then washed twice in stainingbuffer then immersed in 0.1% Triton-X (Sigma-Aldrich). Intracellularstaining was performed using anti-granzyme-B AF647 (eBiosciences) at1:400 in staining buffer for 1 hr at room temperature away from light.Following another two washes in staining buffer, actin staining wasperformed using Phalloidin-Texas Red X (Invitrogen) at a ratio of 1:200in PBS and incubated for 1 hr at room temperature away from light.Finally, the samples were washed in PBS twice, and a drop of Vectashieldwith DAPI (USB Corp.) was added to a microscope slide before mountingthe coverslip.

Cell-Mediated Cytotoxicity

Lactate dehydrogenase (LDH) release assay (Roche, Penzberg, Germany) wasused to measure CD8⁺ T cell lytic activity against B16F10-OVA targetcells in vitro according to the manufacturer's guidelines. Briefly,B16F10-OVA cells were cultured in DMEM media with 10% FBS and 2%penicillin. After confluence, cells were dissociated using trypsin EDTA(Invitrogen). A total of 1·10⁴ target cells were incubated with seriallydiluted, previously activated effector T cells in 200 μl assay medium(cell culture media with 2% FBS) in a 96-well plate. After 4 hr, theplate was centrifuged at 1500 rpm, the supernatant was removed, and 100μl of LDH reaction mixture was added to cells. The plate was incubatedat room temperature away from light for 10 min, and absorbance wasmeasured at 492 nm. Percent cell-mediated cytotoxicity was calculated asfollows:

100×(experimental−effector spontaneous−target spontaneous)/(targetmaximum−target spontaneous).

Results

The efficacy of CNPs as an aAPC was examined by incubating the CNPs fromExample 6 with CD8⁺ T cells isolated from an OT-1 mouse. FIG. 18compares expansion as a function of time for T cells interacting withCNPs; ^(N)CNTs, Dynabeads® (commercially available magnetic beads), orsoluble tetramers presenting antigens with exogenous IL-2 (^(N)CNT-EXO,DYNA-EXO and TET-EXO respectively); ^(N)CNTs, Dynabeads®, or solubletetramers presenting antigens without the addition of IL-2 (^(N)CNT,DYNA and TET respectively). After two weeks, CD8⁺ T cells expanded abouttwo hundred-fold, more than twice the cumulative expansion of cellsinteracting with DYNA-EXO, and more than four times the results obtainedfrom TET-EXO. Sizing of cells interacting with these same platformsindicates that these effects on expansion potentially extend to cellphenotype (Table 7). Finally, measurement of IFN-gamma released fromthese CD8⁺ T cells parallels previous results on cell proliferation andsize (FIG. 19).

Confocal imaging indicated that early interaction of CD8⁺ T cells withCNPs is characterized by the formation of non-specific cell blastsaround the particles and with MHC-I loaded with a null agonist peptide(SIYRYYGL) that does not trigger the OT-1 T cell receptor. Upregulationof effector markers was however observed in T cells interacting withCNPs presenting OT-1-specific antigens. It was characterized withconfocal imaging by the presence of cytotoxic granules and by thesecretion of granzyme-B. Expression of CD25, the a-chain of the IL-2receptor, was also observed at the T cell-CNP interphase. As expected, Tcells cultured in the presence of CNPs presenting MHC-I loaded withSIYRYYGL peptide did not express CD25 or granzyme-B. T cells incubatedwith soluble tetramers expressed these effector markers, although not tothe same level of fluorescence intensity as what was observed with CNPs.T cell cultures with CNPs displaying agonist peptide were observed withan inverted-phase contrast microscope at 24, 48, and 72 hours. Thecultures showed the formation of large lymphoblasts around the particleat 24 hours, and increasing in size through 48 and 72 hourscharacteristic of vigorous T cell proliferation.

TABLE 7 Effect of CNP on T cell proliferation and cell size as afunction of time Cell Fold Diameter Groups Days Expansion (μm) CNP 3 7.0 ± 0.1 9.3 ± 0.1 5 22.4 ± 0.9 9.2 ± 0.1 7 65.8 ± 5.2 8.1 ± 0.1 14191.9 ± 15.9 7.6 ± 0.0 TET-EXO 3  7.1 ± 0.2 8.6 ± 0.0 5 17.7 ± 0.5 7.6 ±0.0 7 37.0 ± 1.3 6.5 ± 0.2 14 44.1 ± 3.6 6.7 ± 0.0 DYNA-EXO 3  7.3 ± 0.28.8 ± 0.1 5 20.8 ± 0.6 7.9 ± 0.0 7 46.7 ± 1.9 6.8 ± 0.1 14 77.1 ± 3.67.1 ± 0.1

Flow cytometry analysis of activated cell phenotype indicated that ahigher percentage of CD8⁺ T cells expanded using CNPs retainedexpression of CD27—a marker of T cell expansion, CD69—an earlyactivation marker, the IL-2 receptor, CD25, and CD62L—an L-selectinadhesion receptor on naïve and central memory T cells. FIG. 20 is a plotof the percentage of CD8⁺/CD27⁺ T cells by group as a function of time.The percentage of CD8⁺/CD27⁺ T cells activated by CNPs was consistentlyabove 90% during the first week of culture, and was significantly higherthan the percentage of CD8⁺/CD27⁺ T cells generated by DYNA-EXO andTET-EXO at day 5, 7 and 14. In addition, at least 90% of CD8⁺ T cellsactivated by CNPs were CD69⁺/CD25⁺ in our culture conditions (see FIG.21); this portion of cell population was at a significantly largerpercentage than what was generated by other platforms at day 5 andthereafter. Furthermore, the percentage of CD8⁺ T cells expressing aCD44⁺/CD62L⁺ phenotype was the highest in the CNP group (See FIG. 22)).Overall, T cells activated by CNPs were able to retain theirproliferative capacity and effector phenotype better than controlplatforms. In addition, expression of granzyme-B in T cells activated byCNPs was significantly higher than lymphocytes cultured with DYNA-EXOand TET-EXO. This is demonstrated in FIG. 23 showing the expression ofintracellular granzyme-B at day 3 in OT-1 CD8⁺ T cells activated by CNPas compared to controls. FIG. 24 shows the overall cellular toxicity ofOT-1 CD8⁺ T cells towards B16 cells presenting MHC-I in the context ofOVA. Cell-specific cytolytic activity of lymphocytes previously expandedwith CNPs and cultured with a melanoma tumor cell line (B16-OVA) wassignificantly higher compared to other platforms. At E:T ratio of 20:1,specific lysis of target tumor cells was measured ˜60% for T cellsexpanded by CNPs; as much as three times the cytolytic activity observedin cells expanded using conventional, commercially available magneticbeads supplemented with IL-2 in culture (DYNA-EXO).

In vitro stimulation with CNPs requires a thousand-fold less IL-2

Previous work has established the importance of IL-2 in increasing theproliferative capacity of T cells. Importantly, a recent study hasdemonstrated that at least a thousand-fold higher exogenous IL-2concentrations is required to match the effects of sustained paracrinedelivery. See Steenblock et al. (2011), J. Biological Chem.286:34883-34892 and Labowsky and Fahmy (2012), Chem. Eng. Sci.74:114-123. For this reason, we chose to approximate the observedeffects of CNPs on CD8⁺ T cells by adding a thousand-fold more exogenousIL-2 in control groups, and test the therapeutic efficacy of activated Tcells in the context of a murine B16 melanoma model. Flow cytometryanalysis of the expression of CD69, CD25, CD44 and CD62L expressionsfrom CD8⁺ T cells cultured for 3 days with either CNPs, Dynabeads® witha thousand-fold more IL-2 (DYNA-EXO¹⁰⁰⁰), and soluble tetramers with athousand-fold more IL-2 (TET-EXO¹⁰⁰⁰) revealed no significantdifferences in the percentage of activated T cell populations across thethree groups. Control groups significantly unregulated the expression ofT cell activation markers, especially CD25 and CD69 when compared toprevious day 3 measurements using a thousand-fold less IL-2. FIG. 25shows the expression of intracellular granzyme-B at day 3 in OT-1 CD8⁺ Tcells activated by CNP vs. DYNA-EXO¹⁰⁰⁰ and TET-EXO¹⁰⁰⁰. In the contextof T cell function, intracellular granzyme-B levels increased for Tcells previously cultured with DYNA-EXO¹⁰⁰⁰ and TET-EXO¹⁰⁰⁰ (by as muchas 55% for the latter) as compared to an absence of a thousand-foldexcess IL-2. This was found to be consistent with previous studiesshowing that IL-2 increases the cytolytic function in a dose-dependentmanner during the primary activation of murine CD8+ T cells in vitro.FIG. 26 depicts the cellular cytotoxicity of OT-1 CD8⁺ T cells towardsB16 cells presenting MHC-I in the context of OVA, comparing T cellsactivated by CNP vs. DYNA-EXO¹⁰⁰⁰ and TET-EXO¹⁰⁰⁰. The observed increasein intracellular granzyme-B for T cells cultured using control groupsresulted in a significant enhancement in cell-specific cytolyticactivity. For instance, specific lysis of target tumor cells at an E:Tratio of 20:1 was found to be similar in CD8⁺ T cells previouslycultured with CNPs, DYNA-EXO¹⁰⁰⁰, and TET-EXO¹⁰⁰⁰.

Example 8. Adoptive Immunotherapy with CNP Expanded T Cells in MurineMelanoma Model

Materials and Methods

In Vivo B16 Melanoma Study

C57BL/6 Mice were accommodated in autoclaved micro-isolator cages thatwere housed in a positive pressure containment rack, and maintainedunder the guidelines of an approved protocol from the Yale UniversityInstitutional Animal Care and Use Committee. Mice were randomly assignedto groups of six animals each. The xenografts of melanoma were developedby subcutaneously implanting 5×10⁶ B16F10-OVA in the right flank of themice. After 10 days of tumor inoculation, each mouse was treated withactivated OT-1 CD8⁺ T cells by direct injection into the tumor. Thetumor inhibition activity was determined with the tumor volume, whichwas calculated by the following equation: V=(w)²×(l)/2, where (w) and(l) were the width and length of the tumor as measured by a caliper.Animals were sacrificed when they met any of the following conditions:(1) 15% loss in initial body weights (2) the size of the tumor 1.5 cm inany dimension, (3) the mouse became lethargic, sick or unable to feed,(4) the mouse developed an ulcerated tumor.

Isolation of Tumor Infiltrating Lymphocytes (TIL)

Subcutaneous B16 melanoma tumors were resected from mice, weighed,minced, and then placed in a serum-free RPMI media containing 175 U/mLof Collagenase IA (Sigma-Aldrich). The resulting tissue suspension wasincubated at 37° C. for 1 hr, passed through a 70-μm tissue filter, andthe resulting cells were washed twice in serum free RPMI media. Thepellet was re-suspended in 0.5 mL of RPMI media then overlaid over mouselympholyte-M media (Accurate Chemical, Westbury, N.Y.) for lymphocyteisolation, followed by centrifugation at 1500 rpm per the manufacturer'sinstructions. The resulting buffy coat layer was removed and washed inRPMI media and subsequently re-suspended in 1 mL of staining buffer. Allcell suspensions were counted to determine absolute numbers of isolatedTILs and subsequently distributed to FACS tube for FACS staining andanalysis.

Immunohistochemistry

Resected tumor tissue was fixed in 10% formalin for 24 hrs. and embeddedin paraffin. Preparation of slides, sectioning, andhaematoxylin-and-eosin (H&E) staining were performed by the Yalehistology and pathology laboratory.

Results

We evaluated the effects of CNPs from Example 6 for adoptive therapyagainst an aggressive tumor model, the B16 mouse melanoma model, bytransferring CD8⁺ T cells via a single peritumoral injection. Mice wereinoculated with the B16 tumor for ten days before T cell injection. Asignificant delay in tumor growth at day 14 can be observed with animalsadoptively transferred with CNP-cultured T cells compared to thosewithout any treatment. Similar therapeutic effects with other platformssuch as Dynabeads®, can be achieved but only with a thousand-fold moreIL-2 (FIGS. 27 and 28). To elucidate the immunologic mechanisms behindthe delayed tumor growth with CNP-cultured T cells, tumor-infiltratinglymphocytes (TILs) were harvested from the tumors of animals euthanizedat day 14. Both the quantity and quality of TILs in animals adoptivelytransferred with CNP-cultured T cells were significantly different. Ahigher count of CD8⁺ T cells was detected in the tumor microenvironment,as indicated by the absolute number of isolated TILs per tumor in micetreated with CNP-stimulated T cells versus control platforms (FIG. 29).The high count of tumor-infiltrating CD8⁺ T cells in CNP-T cell culturedmice was measured to be similar to mice treated with T cells previouslyexpanded using DYNA-EXO¹⁰⁰⁰, but at least an order of magnitude higherthan other control groups. TILs isolated from tumors treated with CNP orDYNA-EXO¹⁰⁰⁰ were terminally differentiated into effectors as indicatedby the high proportion of cells expressing a CD44⁺/CD62L⁻ phenotype(FIG. 30). Histological evaluation of tumor tissue in the CNP groupconfirms evidence of lymphocyte infiltration, and apoptosis in the tumorcells. This was also observed in tumor tissues isolated fromDYNA-EXO¹⁰⁰⁰ mice, but to a lesser extent in the tissues isolated fromTET-EXO¹⁰⁰⁰ mice. As expected, tissue samples from tumors in micereceiving no treatment showed the highest extent of cytologicpolymorphism. One notable difference was also the decrease inmicrovessel density observed in samples isolated from the CNPs andDYNA-EXO¹⁰⁰⁰; this was in addition to a decrease in tumor cell densityas observed in the hematoxylin and eosin stain of tumor samples frommice euthanized at the end of the study. This decrease in tumor cellproliferation is consistent with the lack of new vasculature needed tosupply the growing tumor mass.

1.-17. (canceled)
 18. A method for adoptive immunotherapy of a diseaseor disorder characterized by over-activation, undesirable or aberrantactivation of an immune response comprising isolating a population ofCD4⁺CD45⁺ T cells from a subject to be treated, activating a populationof CD4⁺CD45⁺ T cells isolated from a subject with a nanotube particlecomposite comprising carbon nanotubes, the nanotubes having bound to orpresent on the surface one or more T cell receptor activators andnanoparticles comprising an immunostimulatory agent, expanding theCD4⁺CD45⁺ T cells, and administering the CD4⁺CD45⁺ T cells to thesubject to be treated in an amount effective to eliminate or reduce therisk or delay the outset of conditions associated with undesirableactivation, over-activation or inappropriate or aberrant activation ofan immune response.
 19. The method of claim 18, wherein the disease ordisorder is selected from the group consisting of allergic disease,autoimmune diseases or disorders, graft rejection or graft-versus-hostdisease.
 20. The method of claim 18, wherein the nanotube particlecomposite further comprises a magnetic particle, and wherein the methodfurther comprises separating the CD4⁺CD45⁺ T cells from the nanotubeparticle composite prior to administering them to the subject to betreated. 21.-22. (canceled)